Methods, compounds, and compositions for reducing body fat and modulating fatty acid metabolism

ABSTRACT

Methods, pharmaceutical compositions, and compounds for reducing body weight, modulating body lipid metabolism, and reducing food intake in mammals are provided. The compounds of the invention include fatty acid ethanolamide compounds, homologues and analogs of which the prototype is the endogenous fatty acid ethanolamide, oleoylethanolamide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Patent Application No.60/336,289 filed Oct. 31, 2001 and U.S. Patent Application 60/279,542filed Mar. 27, 2001. The contents of which are each incorporated hereinby reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. DA12653, awarded by the National Institute of Health. The Government hascertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to fatty acid ethanolamides, their homologues,and their analogs and to their use as pharmacologically active agents toreduce body fat, reduce food consumption, and modulate lipid metabolism.

BACKGROUND OF THE INVENTION

Obesity is a worldwide health challenge occurring at alarming levels inthe United States and other developed nations. About 97 million adultsin the United States are overweight. Of these 40 million are obese.Obesity and overweight greatly increase the risk of many diseases.Hypertension; type 2 diabetes; dyslipidemia; coronary heart disease;stroke; gallbladder disease; osteoarthritis; sleep apnea and otherrespiratory problems; and endometrial, breast, prostate, and coloncancers have been associated with higher body weights. Persons withhigher body weights also suffer from a higher all-cause death rate.According to the National Institutes of Health about 280,000 adultdeaths in the United States each year may be attributed in part toobesity.

Weight loss is desirable in the case of obesity and overweightindividuals. Weight loss can help to prevent many of these harmfulconsequences, particularly with respect to diabetes and cardiovasculardisease (CVD). Weight loss may also reduce blood pressure in bothoverweight hypertensive and non-hypertensive individuals; serumtriglycerides levels and increases the beneficial high-densitylipoprotein (HDL)-form of cholesterol. Weight loss also generallyreduces somewhat the total serum cholesterol and low-density lipoprotein(LDL)-cholesterol levels. Weight loss may also reduce blood glucoselevels in overweight and obese persons.

While weight loss is desirable, it is hard to achieve. Many treatmentsfor the management of overweight and obesity and the maintenance ofweight loss exist. However, recidivism is rampant. Approximately, 40percent of women and 24 percent of men are trying to actively loseweight at any given time. These treatments include low-calorie diets andlow-fat diets; increased physical exercise; behavioral therapiesdirected toward reducing food intake, pharmacotherapy; surgery; andcombinations of the above.

The pharmacopoeia of weight loss is relatively bare. Drugs such assibutramine, dexfenfluramine, orlistat, phenylpropanolamine,phenteramine, or fenfluramine can facilitate weight loss in obese adultswhen used for prolonged periods. In general, however, the safety oflong-term administration of pharmaco-therapeutic weight loss agents isunknown. For instance, recently due to concerns about valvular heartdisease observed in patients, fenfluramine and dexfenfluramine have beenwithdrawn from the market. In the face of the slim pharmacopoeia and thehigh prevalence of obesity and overweight, there is a need for newpharmaceutical methods and compositions to promote and maintain weightloss.

Fatty acid ethanolamides (FAE) are unusual components of animal andplant lipids, and their concentrations in non-stimulated cells aregenerally low (Bachur et al., J. Biol. Chem., 240:1019-1024 (1965);Schmid et al., Chem. Phys. Lipids, 80:133-142 (1996); Chapman, K. D.,Chem. Phys. Lipids, 108:22 1-229 (2000)). FAE biosynthesis can berapidly enhanced, however, in response to a wide variety ofphysiological and pathological stimuli, including exposure to fungalpathogens in tobacco cells (Chapman et al., Plant Physiol.,116:1163-1168 (1998)), activation of neurotransmitter receptors in ratbrain neurons (Di Marzo et al., Nature, 372:686-691 (1994); Giuffrida etal., Nat. Neurosci., 2:358-363 1999)) and exposure to metabolicstressors in mouse epidermal cells (Berdyshev et al., Biochem. J,346:369-374 (2000)). The mechanism underlying stimulus-dependent FAEgeneration in mammalian tissues is thought to involve two concertedbiochemical reactions: cleavage of the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by an unknown phospholipaseD; and NAPE synthesis, catalyzed by a calcium ion- and cyclicAMP-regulated N-acyltransferase (NAT) activity (Di Marzo et al., Nature,372:686-691 (1994); Cadas et al., J NeuroSci., 6:3934-3942 (1996); Cadaset al., H., J. Neurosci., 17:1226-1242.(1997)).

The fact that both plant and animal cells release FAEs in astimulus-dependent manner suggests that these compounds may playimportant roles in cell-to-cell communication. Further support for thisidea comes from the discovery that the polyunsaturated FAE, anandamide(arachidonylethanolamide), is an endogenous ligand for cannabinoidreceptors (Devane et al., Science, 258:1946-1949 (1992))—Gprotein-coupled 12 receptors expressed in neurons and immune cells,which recognize the marijuana constituent Δ⁹-tetrahydrocannabinol(Δ⁹-THC) (for review, see reference (Pertwee, R. G., Exp. Opin. Invest.Drugs, 9:1553-1571 (2000)).

Two observations make it unlikely that other FAEs also participate incannabinoid neurotransmission. The FAE family is comprised for the mostpart of saturated and monounsaturated species, such aspalmitylethanolamide and oleoylethanolamide, which do not significantlyinteract with cannabinoid receptors (Devane et al., Science,258:1946-1949 (1992); Griffin et al., J. Pharmacol. Exp. Ther.,292:886-894. (2000)). Second, when the pharmacological properties of theFAEs have been investigated in some detail, as is the case withpalmitylethanolamide, such properties have been found to differ fromthose of Δ⁹-THC and to be independent of activation of known cannabinoidreceptor subtypes (Calignano et al., Nature, 394:277-281 (1998)). Thus,the biological significance of the FAEs remains elusive.

Oleoylethanolamide (OEA) is a natural analogue of the endogenouscannabinoid anandamide. Like anandamide, OEA is produced in cells in astimulus-dependent manner and is rapidly eliminated by enzymatichydrolysis, suggesting a role in cellular signaling. However, unlikeanandamide, OEA does not activate cannabinoid receptors and itsbiological functions were hereto-fore essentially unknown.

There is a need for additional methods and agents to treat obesity andoverweight as well as to maintain weight loss. The present inventionmeets this need by providing novel methods and pharmaceuticalcompositions related to our instant discovery that oleoylethanolamide(OEA) and other fatty acid ethanolamide compounds (e.g.,palmitylethanolamide, elaidylethanolamide)) can reduce appetite, foodintake, body weight, and body fat and alter fat metabolism.

SUMMARY OF THE INVENTION

The present invention provides compounds, compositions, and methods forreducing body fat and for treating or preventing obesity, and overweightin mammals and the diseases associated with these health conditions. Inone aspect, the invention provides methods for reducing body fat or bodyweight and for treating or preventing obesity or overweight and forreducing food intake by administration of pharmaceutical compositionscomprising a fatty acid alkanolamide compound, homologue, or analog inan amount sufficient to reduce body fat, body weight or prevent body fator body weight gain. In other aspects, the invention is drawn to thefatty acid ethanolamide compounds, homologues, analogs; and theirpharmaceutical compositions and such methods of use.

In other embodiments, the fatty acid moiety of the fatty acidalkanolamide or ethanolamide compound, homologue, or analog may besaturated or unsaturated, and if unsaturated may be monounsaturated orpolyunsaturated.

In some embodiments, the fatty acid moiety of the fatty acidalkanolamide compound, homologue, or analog is a fatty acid selectedfrom the group consisting of oleic acid, palmitic acid, elaidic acid,palmitoleic acid, linoleic acid, alpha-linolenic acid, andgamma-linolenic acid. In certain embodiments, the fatty acid moietieshave from twelve to 20 carbon atoms.

Other embodiments are provided by varying the hydroxyalkylamide moietyof the fatty acid amide compound, homologue or analog. These embodimentsinclude the introduction of a substituted or unsubstituted lower (C₁-C₃)alkyl group on the hydroxyl group of an alkanolamide or ethanolamidemoiety so as to form the corresponding lower alkyl ether. In anotherembodiment, the hydroxy group of the alkanolamide or ethanolamide moietyis bound to a carboxylate group of a C₂ to C₆ substituted orunsubstituted alkyl carboxylic acid to form the corresponding ester ofthe fatty acid ethanolamide. Such embodiments include fatty acidalkanolamide and fatty acid ethanolamides in ester linkage to organiccarboxylic acids such as acetic acid, propionic acid, and butanoic acid.In one embodiment, the fatty acid alkanolamide is oleoylalkanolamide. Ina further embodiment, the fatty acid alkanolamide is oleoylethanolamide.

In still another embodiment, the fatty acid ethanolamide compound,homologue, or analog further comprises a substituted or unsubstitutedlower alkyl (C₁-C₃) group covalently bound to the nitrogen atom of thefatty acid ethanolamide.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable excipient and a compound, orits pharmaceutically acceptable salt, having the formula:

In this formula, n is from 0 to 5 and the sum of a and b can be from 0to 4. Z is a member selected from C(O)N(R^(o))—; —(R^(o))NC(O)—;—OC(O)—; —(O)CO—; O; NR^(o); and S, in which R¹ and R² are independentlyselected from the group consisting of unsubstituted or unsubstitutedalkyl, hydrogen, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted lower (C₁-C₆) acyl, homoalkyl, and aryl. Up to fourhydrogen atoms of either or both the fatty acid portion and ethanolamineportion of the compound may also be substituted by methyl or a doublebond. In addition, the molecular bond between carbons c and d may beunsaturated or saturated. In some embodiments, the fatty acidethanolamide of the above formula is a naturally occurring compound.

In other aspects of the invention, the methods and compositions employfatty acid ethanolamide and fatty acid alkanolamide compounds, homologsand analogs for reducing body weight in which the compounds, homologsand analogs cause weight loss when administered to test animals (e.g.,rats, mice, rabbits, hamsters, guinea pigs).

In still other aspects, the invention is drawn to methods of usingarylthiazolidinedione compounds and heteroaryl and aryl oxyacetic acidtype compounds to reduce body fat, body weight and appetite.

Still other aspects of the invention address methods of using andadministering the subject compounds and compositions for reducing bodyweight or reducing appetite or reducing food intake or causinghypophagia in mammals (e.g., humans, cats or dogs). The subjectcompositions may be administered by a variety of routes, includingorally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Starvation increases circulating oleoylethanolamide levels inrats: (a) time course of the effects of food deprivation on plasmaoleoylethanolamide (oleylethaolamide, OEA) levels; (b) effect of waterdeprivation (18 h) on plasma oleoylethanolamide levels; (c) effect offood deprivation (18 h) on oleoylethanolanide levels in cerebrospinalfluid (CSF); (d) time course of the effects of food deprivation onplasma anandamide (arachidonylethanolamide, AEA) levels; (e) effect ofwater deprivation (18 h) on anandamide plasma levels; (f) effect of fooddeprivation (18 h) on anandamide levels in CSF. Results are expressed asmean±s.e.m.; asterisk, P<0.05; two asterisks, P<0.01, n=10 per group.

FIG. 2. Adipose tissue is a primary source of circulatingoleoylethanolamide: starvation-induced changes in N-acyltransferase(NAT) and fatty acid amide hydrolase (FAAH) activities in various rattissues. (a) fat; (b) brain; (c) liver; (d) stomach; (e) smallintestine. Empty bars, free-feeding animals; filled bars, 18-h fastedanimals. Activities are in pmol/mg protein/min. Asterisk, P<0.05, n=3.

FIG. 3. Adipose tissue is a primary source of circulatingoleoylethanolamide: starvation-induced changes in NAPE andoleoylethanolamide (oleoylethanolamide, OEA) content in adipose andliver tissues. (a) structures of the oleoylethanolamide precursorsalk-1-palmitoenyl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-oleyl(left panel, NAPE 1) andalk-1-palmityl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-oleyl(right panel, NAPE 2); (b) representative HPLC/MS tracings for selectedions characteristic of NAPE 1 (left panel, m/z=987, deprotonatedmolecule, [M-H]⁻) and NAPE 2 (right panel, m/z=1003, [M-H]⁻) infree-feeding (top) and 18-h fasting rats (bottom); (c) food deprivation(18 h) increases the content of NAPE species in fat and decreases it inliver. All identifiable NAPE species were quantified, including theoleoylethanolamide precursors NAPE1 and NAPE 2, and the PEA precursorNAPE 3; (d) food deprivation (18 h) increases oleoylethanolamide contentin fat and liver. Empty bars, free-feeding animals; filled bars, 18-hfasted animals. Asterisk, P<0.05, Student's t test; n=3.

FIG. 4. Oleoylethanolamide/pranamide selectively suppresses food intake:(a) dose-dependent effects of oleoylethanolamide(oleoylethanolamide/OEA/pranamide) (i.p., empty squares),elaidylethanolamide (empty circles), PEA (triangles), oleic acid (filledsquares) and anandamide (filled circles) on food intake in 24-hfood-deprived rats. Vehicle alone (70% DMSO in saline, 1 ml per kg,i.p.) had no significant effect on acute food intake; (b) time course ofthe hypophagic effects of oleoylethanolamide (20 mg per kg, i.p.)(squares) or vehicle (lozenges) on food intake. (c) effects of vehicle(V), lithium chloride (LiCl, 0.4 M, 7.5 ml per kg) or oleoylethanolamide(20 mg per kg) in a conditioned taste aversion assay. Empty bars, waterintake; filled bars, saccharin intake. Effects of vehicle (V) oroleoylethanolamide (5 or 20 mg per kg) on: (d) water intake (expressedin ml per 4 h); (e) body temperature; (f) latency to jump in the hotplate analgesia test; (g) percent time spent in open arms in theelevated plus maze anxiety test; (h) number of crossings in the openfield activity test; (i) number of operant responses for food. Asterisk,P<0.05, n=8-12 per group.

FIG. 5. Effects of subchronic oleoylethanolamide administration on foodintake and body weight: (a) effects of oleoylethanolamide(oleoylethanolamide, OEA) (5 mg per kg, i.p. once a day) (empty bars) orvehicle (5% Tween 80/5% propyleneglycol in sterile saline; filled bars)on cumulative food intake; (b) time course of the effects ofoleoylethanolamide (triangles) or vehicle (squares) on body weightchange; (c) effects of oleoylethanolamide or vehicle on net body weightchange; (d) effects of oleoylethanolamide (5 mg per kg) or vehicle oncumulative water intake. Asterisk, P<0.05; two asterisks, P<0.01, n=10per group.

FIG. 6. Role of peripheral sensory fibers in oleoylethanolamide-inducedanorexia. Effects of vehicle (V), oleoylethanolamide(oleoylethanolamide/pranamide/OEA) (5 mg per kg, i.p.), CCK-8 (10 μg perkg) and CP-93129 (1 mg per kg), a centrally active 5-HT1B receptoragonist, on food intake in a, control rats and c, capsaicin-treatedrats. Water intake in (b) control rats and (d) capsaicin-treated rats.Asterisk, P<0.05; n=8-12 per group.

FIG. 7. Oleoylethanolamide/pranamide increases c-fos mRNA expression indiscrete brain regions associated with energy homeostasis and feedingbehavior: (a) pseudocolor images of film autoradiographs show thatoleoylethanolamide (right section) elicits a striking and selectiveincrease in c-fos mRNA labeling in the paraventricular (PVN) andsupraoptic (SO) hypothalamic nuclei, as assessed by in situhybridization. A representative section from a vehicle-treated rat isshown at left. Labeling densities are indicated by color:blue<green<yellow<red. (b) quantification of c-fos cRNA labeling inforebrain regions [PVN, SO, arcuate (Arc), layer II piriform cortex(pir), ventrolateral thalamas (VI) and S1 forelimb cortex (S1FL)] ofrats treated with vehicle, oleoylethanolamide and oleic acid; (c) filmautoradiogram showing elevated ³⁵S c-fos mRNA expression in the nucleusof the solitary tract (NST) in an oleoylethanolamide-treated rat; Inset,c-fos cRNA labeling in the NST (shown in red) was identified by itslocalization relative to adjacent efferent nuclei (hypoglossal anddorsal motor nucleus of the vagus), which express choline acetyltransferase (ChAT) mRNA (shown in purple); (d) oleoylethanolamideincreases c-fos mRNA expression in NST but not in the hypoglossalnucleus (HgN). Two asterisks, P<0.0001, n=5 per group.

FIG. 8. The effects of OEA, Oleic acid (OA), AEA, PEA, and methyl-OEA onfatty acid oxidation in soleus muscle.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the surprising discovery that OEA and otherfatty acid alkanolamide compounds act to reduce food intake, bodyweight, and body fat and to modulate fatty acid oxidation. It has beensurprisingly discovered that oleoylethanolamide (OEA), a natural lipidof heretofore unknown biological function in mammals, is a potent bodyfat reducing and weight control compound when administered to testanimals. U.S. Patent Application 60/279,542, filed Mar. 27, 2001, andassigned to the same assignee and herein incorporated by reference inits entirety discloses OEA and OEA-like compounds as agents which canreduce body fat and appetite in mammals.

Upon the discovery of the prototype OEA, other fatty acid alkanolamidecompounds and homologs were also found to be active.

OEA can serve as a model in the development of other fatty acidalkanolamide-like fat reducing compounds for treating obesity, inducingweight loss, reducing appetite, or food intake. This invention providessuch other compounds as disclosed below.

The discovery that OEA administration acts to reduce appetite, foodintake, and body weight can be used to identify other fatty acidethanolamides, homologues, and analogs as weight and appetite controlagents. This invention provides such agents.

DEFINITIONS

The abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a compound of the present invention and apharmaceutically acceptable carrier. The term “pharmaceuticalcomposition” indicates a composition suitable for pharmaceutical use ina subject, including an animal or human. A pharmaceutical compositiongenerally comprises an effective amount of an active agent and apharmaceutically acceptable carrier.

Compounds of the invention may contain one or more asymmetric centersand can thus occur as racemates and racemic mixtures, singleenantiomers, diastereomeric mixtures and individual diastereomers. Thepresent invention is meant to comprehend all such isomeric forms of theinventive compounds.

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and Zgeometric isomers.

Some of the compounds described herein may exist with different pointsof attachment of hydrogen, referred to as tautomers. Such an example maybe a ketone and its enol form known as keto-enol tautomers. Theindividual tautomers as well as mixture thereof are encompassed by theinventive formulas.

Compounds of the invention include the diastereoisomers of pairs ofenantiomers. Diastereomers for example, can be obtained by fractionalcrystallization from a suitable solvent, for example methanol or ethylacetate or a mixture thereof. The pair of enantiomers thus obtained maybe separated into individual stereoisomers by conventional means, forexample by the use of an optically active acid as a resolving agent.

Alternatively, any enantiomer of an inventive compound may be obtainedby stereospecific synthesis using optically pure starting materials orreagents of known configuration

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

“Alkanol,” as used herein refers to a saturated or unsaturated,substituted or unsubstituted, branched or unbranched alkyl group havinga hydroxyl substituent, or a substituent derivable from a hydroxylmoiety, e.g., ether, ester. The alkanol is preferably also substitutedwith a nitrogen-, sulfur-, or oxygen-bearing substituent that isincluded in bond Z (Formula I), between the “fatty acid” and thealkanol.

“Fatty acid,” as used herein, refers to a saturated or unsaturatedsubstituted or unsubstituted, branched or unbranched alkyl group havinga carboxyl substituent. Preferred fatty acids are C₄-C₂₂ acids. Fattyacid also encompasses species in which the carboxyl substituent isreplaced with a —CH₂— moiety.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” includes both aryl and heteroaryl rings asdefined above. Thus, the term “arylalkyl” is meant to include thoseradicals in which an aryl group is attached to an alkyl group (e.g.,benzyl, phenethyl, pyridylmethyl and the like) including those alkylgroups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂in a number ranging from zero to (2m′+1), where m′ is the total numberof carbon atoms in such radical. R′, R″, R′″ and R“ ” each preferablyindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ andCH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are preferably independently selected from hydrogen, (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl and heteroaryl, (unsubstitutedaryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R“ ” groups when more than one of these groups is present.

The term “body fat reduction” means loss of a portion of body fat.

The formula for Body Mass Index (BMI) is [Weight in pounds÷Height ininches÷Height in inches]×703. BMI cutpoints for human adults are onefixed number, regardless of age or sex, using the following guidelines:Overweight human adults individuals have a BMI of 25.0 to 29.9. Obesehuman adults have a BMI of 30.0 or more. Underweight adults have a BMIless of than 18.5. A normal body weight range for an adult is defined asa BMI between 18.5 and 25. BMI cutpoints for children under 16 aredefined according to percentiles: Overweight is defined as a BMI for agegreater than ≧85th percentile and obesity is defined as a BMI-for-age≧95th percentile. Underweight is a BMI-for-age <5th percentile. A normalbody weight range for a child is defined as a BMI above the 5thpercentile and below the 85 percentile.

The term “fatty acid oxidation” relates to the conversion of fatty acids(e.g., oleate) into ketone bodies.

The term “hepatocytes” refers to cells originally derived from livertissue. Hepatocytes may be freshly isolated from liver tissue orestablished cell lines.

The term “modulate” means to induce any change including increasing ordecreasing. (e.g., a modulator of fatty acid oxidation increases ordecreases the rate of fatty oxidation.

The term “muscle cells” refers to cells derived from the predominantcells of muscle tissue. Muscle cells may be freshly isolated from muscletissue or established cell lines.

The term “obese” indicates a body weight 20% over ideal body weight asmeasured by body mass index

Oleoylethanolamide (OEA) refers to a natural lipid of the followingstructure:

In the formulas herein, “Me” represents the methyl group.

The term “weight loss” refers to loss of a portion of total body weight.

The term “pharmaceutically acceptable carrier” encompasses any of thestandard pharmaceutical carriers, buffers and excipients, includingphosphate-buffered saline solution, water, and emulsions (such as anoil/water or water/oil emulsion), and various types of wetting agentsand/or adjuvants. Suitable pharmaceutical carriers and theirformulations are described in REMINGTIONS PHARMACEUTICAL SCIENCES (MackPublishing Co., Easton, 19th ed. 1995). Preferred pharmaceuticalcarriers depend upon the intended mode of administration of the activeagent. Typical modes of administration are described below.

The term “effective amount” means a dosage sufficient to produce adesired result. The desired result may comprise a subjective orobjective improvement in the recipient of the dosage. A subjectiveimprovement may be decreased appetite or craving for food. An objectiveimprovement may be decreased body weight, body fat, or food, decreasedfood consumption, or decreased food seeking behavior.

A “prophylactic treatment” is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of adisease, wherein treatment is administered for the purpose of decreasingthe risk of developing a pathology associated with increased body weightor body fat. The compounds of the invention may be given as aprophylactic treatment to prevent undesirable or unwanted weight gain.

A “therapeutic treatment” is a treatment administered to a subject whoexhibits signs of pathology, wherein treatment is administered for thepurpose of diminishing or eliminating those pathological signs.

The term “to control weight” encompasses the loss of body mass or thereduction of weight gain over time.

The methods, compounds and compositions of the present invention aregenerally useful for reducing or controlling body fat and body weight inmammals. For instance, the methods, compositions, and compounds of thepresent invention are helpful in reducing appetite or inducinghypophagia in mammals. The methods, compounds, and compositions are alsouseful in preventing or mitigating the diseases associated withoverweight or obesity by promoting the loss of body fat and body weight.

The methods, compositions, and compounds of the present inventioninclude modulators of lipid metabolism, and particularly, fat and fattyacid catabolism.

COMPOUNDS OF THE INVENTION

Certain compounds of the present invention may possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

Such compounds of the invention may be separated into diastereoisomericpairs of enantiomers by, for example, fractional crystallization from asuitable solvent, for example methanol or ethyl acetate or a mixturethereof. The pair of enantiomers thus obtained may be separated intoindividual stereoisomers by conventional means, for example by the useof an optically active acid as a resolving agent.

Alternatively, any enantiomer of such a compound of the invention may beobtained by stereospecific synthesis using optically pure startingmaterials of known configuration.

The compounds of the present invention may have unnatural ratios ofatomic isotopes at one or more of their atoms. For example, thecompounds may be radiolabeled with isotopes, such as tritium orcarbon-14. All isotopic variations of the compounds of the presentinvention, whether radioactive or not, are within the scope of thepresent invention.

The instant compounds may be isolated in the form of theirpharmaceutically acceptable acid addition salts, such as the saltsderived from using inorganic and organic acids. Such acids may includehydrochloric, nitric, sulfuric, phosphoric, formic, acetic,trifluoroacetic, propionic, maleic, succinic, malonic and the like. Inaddition, certain compounds containing an acidic function can be in theform of their inorganic salt in which the counterion can be selectedfrom sodium, potassium, lithium, calcium, magnesium and the like, aswell as from organic bases. The term “pharmaceutically acceptable salts”refers to salts prepared from pharmaceutically acceptable non-toxicbases or acids including inorganic bases or acids and organic bases oracids.

The invention also encompasses prodrugs of the present compounds, whichon administration undergo chemical conversion by metabolic processesbefore becoming active pharmacological substances. In general, suchprodrugs will be derivatives of the present compounds that are readilyconvertible in vivo into a functional compound of the invention.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs”,ed. H. Bundgaard, Elsevier, 1985. The invention also encompasses activemetabolites of the present compounds.

A. Fatty Acid Alkanolamide Compounds, Homologs, and Analogs.

Compounds of the invention include body fat reducing fatty acidalkanolamide compounds, including the fatty acid ethanolamide compounds,and their homologues and certain analogs of the fatty acidalkanolamides. Such compounds may be identified and defined in terms ofeither an ability to cause reduced appetite, food intake, and/or bodyweight or body fat upon administration to test animals in vivo.

A variety of such fatty acid alkanolamides, homologs and analogs aretherefore contemplated. Compounds of the invention include compounds ofthe following general formula:

In this formula, n is from 0 to 5 and the sum of a and b can be from 0to 4. Z is a member selected from —C(O)N(R^(o))—; —(R^(o))NC(O)—;—OC(O)—; —(O)CO—; O; NR^(o); and S, in which R^(o) and R¹ areindependently selected from the group consisting of unsubstituted orunsubstituted alkyl, hydrogen, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted lower (C₁-C₆) acyl, homoalkyl, and aryl. Upto four hydrogen atoms of either or both the fatty acid portion andalkanolamine (e.g. ethanolamine) portion of the compound may also besubstituted by methyl or a double bond. In addition, the molecular bondbetween carbons c and d may be unsaturated or saturated. In someembodiments, the fatty acid ethanolamide of the above formula is anaturally occurring compound.

Compounds of the invention also include compounds of the followingformula:

In one embodiment, the compounds of Formula Ia have n from 0 to 5; and asum of a and b that is from 0 to 4; and members R¹ and R² independentlyselected from the group consisting of hydrogen, substituted orunsubstituted C₁-C₆ alkyl, lower substituted or unsubstituted (C₁-C₆)acyl, homoalkyl, and substituted or unsubstituted aryl. In thisembodiment, up to four hydrogen atoms of the fatty acid portion andalkanolamine (e.g., ethanolamine) portion of compounds of the aboveformula may also be substituted by methyl or a double bond. In addition,the molecular bond between carbons c and d may be unsaturated orsaturated. In some embodiments with acyl groups, the acyl groups may bethe propionic, acetic, or butyric acids and attached via an esterlinkage as R² or an amide linkage as R¹.

In another embodiment, the above compounds particularly include those inwhich the fatty acid moiety comprises oleic acid, elaidic acid, orpalmitic acid. Such compounds include oleoylethanolamide,elaidylethanolamide and palmitylethanolamide

In another embodiment, the compounds of Formula Ia have n from 1 to 3;and a sum of a and b that is from 1 to 3; and members R¹ and R²independently selected from the group consisting of hydrogen,substituted or unsubstituted C₁-C₆ alkyl, and lower substituted orunsubstituted (C₁-C₆) acyl. In this embodiment, up to four hydrogenatoms of the fatty acid portion and alkanolamine (e.g., ethanolamine)portion of compounds of the above formula may also be substituted bymethyl or a double bond. In addition, the molecular bond between carbonsc and d may be unsaturated or saturated. In a further embodiment, themolecular bond between carbons c and d is unsaturated and no otherhydrogen atoms are substituted. In a still further embodiment thereof,the members R¹ and R² are independently selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁-C₃ alkyl, andsubstituted or unsubstituted lower (C₁-C₃) acyl.

Exemplary compounds provide mono-methyl substituted compounds, includingethanolamides, of Formula Ia. Such compounds include:

The methyl substituted compounds of the above formula includeparticularly those compounds where R¹ and R² are both H:(R)1′-methyloleoylethanolamide, S(1′)-methyloleoylethanoiamide,(R)2′-methyloleoylethanolamide, (S)2′-methyloleoylethanolamide, (R)1-methyloleoylethanolamide, and (S) 1-methyloleoylethanolamide.

Reverse OEA-Like Compounds.

Compounds of the invention also include a variety of analogs of OEA.These compounds include reverse OEA compounds of the general formula:

In some embodiments, the invention provides compounds of Formula II.Exemplary the compounds of Formula II have n from 1 to 5, and a sum of aand b from 0 to 4. In this embodiment, the member R² is selected fromthe group consisting of hydrogen, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted lower (C₁-C₆) acyl, homoalkyl, andaryl. In addition, up to four hydrogen atoms of either or both the fattyacid portion and alkanolamine (e.g., ethanolamine) portion of compoundsof the above formula may also be substituted by methyl or a double bond.

Exemplary compounds of formula II include those compounds where thealkanolamine portion is ethanolamine, compounds where R² is H, andcompounds where a and b are each 1, and compounds where n is 1.

One embodiment of a compound according to Formula II is

In another embodiment, the compounds of Formula II have n from 1 to 5and a sum of a and b from 1 to 3. In this embodiment, the member R² isselected from the group consisting of hydrogen, substituted orunsubstituted C₁-C₆ alkyl, and substituted or unsubstituted lower(C₁-C₆) acyl. In addition, up to four hydrogen atoms of either or boththe fatty acid portion and alkanolamine (e.g., ethanolamine) portion ofcompounds of the above formula may also be substituted by methyl or adouble bond.

Oleoylalkanol Ester Compounds.

Compounds of the invention also include oleoylalkanol esters of thegeneral formula:

In some embodiments, the compounds of Formula III, have n from 1 to 5;and the sum of a and b from 0 to 4. The member R² is selected from thegroup consisting of hydrogen, substituted or unsubstituted C₁-C₆ alkyl,lower (C₁-C₆) acyl, homoalkyl, and aryl. Up to four hydrogen atoms ofeither or both the fatty acid portion and alkanol (e.g., ethanol)portion of compounds of the above formula may also be substituted bymethyl or a double bond.

In some embodiments, the compounds of Formula III, have n from 1 to 3;and the sum of a and b from 1 to 3. The member R² is selected from thegroup consisting of hydrogen, substituted or unsubstituted C₁-C₆ alkyl,and substituted or unsubstituted lower (C₁-C₆) acyl. Up to four hydrogenatoms of the fatty acid portion and alkanol (e.g., ethanol) portion ofcompounds of the above formula may also be substituted by methyl or adouble bond.

Compounds of Formula III include those compounds where R² is H,compounds where a and b are each 1, and compounds where n is 1. Examplesof compounds according to Formula III include the oleoyldiethanol ester:

Compounds of Formula III also include mono-methyl substituted oleoylethanol esters such as the (R or S)-2′-methyloleoylethanolesters; the (Ror S)-1′-methyloleoylethanolesters; and the (R orS))-1′-methyloleoylethanolesters; respectively:

Oleoyl Alkanol Ethers

Compounds of the invention also include oleoylalkanol ethers accordingto the general formula:

In some embodiments, the compounds of Formula IV, have an n from 1 to 5and a sum of a and b that can be from 0 to 4. The member R² is selectedfrom the group consisting of hydrogen, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted lower (C₁-C₆) acyl, alkyl, andsubstituted and unsubstituted aryl. Up to four hydrogen atoms of eitheror both the fatty acid portion and alkanol (e.g., ethanol) portion ofcompounds of the above formula may also be substituted by methyl or adouble bond.

In other embodiments, the compounds of Formula IV, have n from 1 to 3;and the sum of a and b can be from 1 to 3. The member R² is selectedfrom the group consisting of hydrogen, substituted or unsubstitutedC₁-C₆ alkyl, and substituted or unsubstituted lower (C₁-C₆) acyl. Up tofour hydrogen atoms of either or both the fatty acid portion and alkanol(e.g., ethanol) portion of compounds of the above formula may also besubstituted by methyl or a double bond.

Compounds of Formula IV include those compounds where R² is H, compoundswhere a and b are each 1, and compounds where n is 1. Examples ofcompounds according to Formula IV include the following (R or S)1′-oleoylethanol ethers and (R or S)-2′-6 oleoylethanol ethers:

Fatty Acid Alkanolamide Analogs Having Polar Head Variants.

Compounds of the invention also include a variety of polar head analogsof OEA. These compounds include compounds having a fatty acid moiety ofthe general formula:

In some embodiments, the compounds of Formula V have a sum of a and bthat can be from 0 to 4. In other embodiments, the sum of a and b isfrom 1 to 3. In these embodiments, up to four hydrogen atoms of thecompounds of the above formula may also be substituted by methyl or adouble bond. In addition, the molecular bond between carbons c and d maybe unsaturated or saturated. A particularly preferred embodiment is thatof the oleic acid fatty acid moiety:

The R³ group of the above structures may be selected from any of thefollowing:

HO—(CH₂)_(z)—NH— wherein z is from 1 to 5, and the alkyl portion thereofis an unbranched methylene chain. For example:

H₂N—(CH₂)_(z)—NH— wherein z is from 1 to 5, and the alkyl portionthereof is an unbranched methylene chain. For example:

HO—(CH₂)_(x)—NH— wherein x is from 1 to 8, and the alkyl portion thereofmay be branched or cyclic. For example,

Additional polar head groups for R³ include, for instance, compoundshaving furan, dihydrofuran and tetrahydrofuran functional groups:

In the above structures, z can be from 1 to 5.

Compounds of the invention include, for instance, those having R polarhead groups based upon pyrrole, pyrrolidine, and pyrroline rings:

In the compounds of the above structures, z can be from 1 to 5.

Other exemplary polar head groups include a variety of imidazole andoxazoles, for example:

In the compounds of the above structures, z can be from 1 to 5.

Oxazolpyridine polar head groups are also exemplary:

Fatty Acid Alkanolamide Analogs Having Apolar Tail Variants.

Compounds of the invention include a variety of alkanolamide andethanolamide compounds having a variety of flexible apolar tails. Thesecompounds include compounds of the following formulas in which Rrepresents an ethanolamine moiety, an alkanolamine moiety, or a stableanalog thereof. In the case of ethanolamine, the ethanolamine moiety isattached preferably via the ethanolamine nitrogen rather than theethanolamine oxygen.

In the above structures, m is from 1 to 9 and p is independently from 1to 5.

An exemplary compound is:

Another exemplary compound is an ethanolamine analog with an apolar tailof the following structural formula:

Exemplary compounds include analogs of fatty acid alkanolamides. Suchanalogs include those compounds taught in U.S. Pat. No. 6,200,998(hereby incorporated by reference). This reference teaches compounds ofthe general formula:

In the above formula, and as defined in U.S. Pat. No. 6,200,998, Ar¹ is(1) arylene or (2) heteroarylene, wherein arylene and heteroarylene areoptionally substituted with from 1 to 4 groups selected from R^(a); Ar²is (1) ortho-substituted aryl or (2) ortho-substituted heteroaryl,wherein said ortho substituent is selected from R; and aryl andheteroaryl are optionally further substituted with from 1-4 groupsindependently selected from R^(a); X and Y are independently O, S,N—R^(b), or CH₂; Z is O or S; n is 0 to 3; R is (1) C₃₋₁₀ alkyloptionally substituted with 1-4 groups selected from halo and C₃₋₆cycloalkyl, (2) C₃₋₁₀ alkenyl, or (3) C₃₋₈ cycloalkyl; R^(a) is (1)C₁₋₁₅ alkanoyl, (2) C₁₋₁₅ alkyl, (3) C₂₋₁₅ alkenyl, (4) C₂₋₁₅ alkynyl,(5) halo, (6) OR^(b), (7) aryl, or (8) heteroaryl, wherein said alkyl,alkenyl, alkynyl, and alkanoyl are optionally substituted with from 1-5groups selected from R^(c) and said aryl and heteroaryl optionallysubstituted with 1 to 5 groups selected from R^(d); R^(b) is (1)hydrogen, (2) C₁₋₁₀ alkyl, (3) C₂₋₁₀ alkenyl, (4) C₂₋₁₀ alkynyl, (5)aryl, (6) heteroaryl, (7) aryl C₁₋₁₅ alkyl, (8) heteroaryl C₁₋₁₅ alkyl,(9) C₁₋₁₅ alkanoyl, (10) C₃₋₈ cycloalkyl, wherein alkyl, alkenyl,alkynyl are optionally substituted with one to four substituentsindependently selected from R^(c), and cycloalkyl, aryl and heteroarylare optionally substituted with one to four substituents independentlyselected from R^(d); or R^(c) is (1) halo, (2) aryl, (3) heteroaryl, (4)CN, (5) NO₂, (6) OR^(f); (7) S(O)_(m)R^(f), m=0, 1 or 2, provided thatR^(f) is not H when m is 1 or 2; (8) NR^(f)R^(f) (9) NR^(f)COR^(f), (10)NR^(f)CO₂R^(f), (11) NR^(f)CON(R^(f))₂, (12) NR^(f)SO₂R^(f), providedthat R^(f) is not H, (13) COR^(f), (14) CO₂R^(f), (15) CON(R^(f))₂, (16)SO₂N(R^(f))₂, (17) OCON(R^(f))₂, or (18) C₃₋₈ cycloalkyl, wherein saidcycloalkyl, aryl and heteroaryl are optionally substituted with 1 to 3groups of halo or C₁₋₆ alkyl; R^(d) is (1) a group selected from R^(c),(2) C₁₋₁₀ alkyl, (3) C₂₋₁₀ alkenyl, (4) C₂₋₁₀ alkynyl, (5) aryl C₁₋₁₀alkyl, or (6) heteroaryl C₁₋₁₀ alkyl, wherein alkyl, alkenyl, alkynyl,aryl, heteroaryl are optionally substituted with a group independentlyselected from R^(e); R^(e) is (1) halogen, (2) amino, (3) carboxy, (4)C₁₋₄ alkyl, (5) C₁₋₄ alkoxy, (6) hydroxy, (7) aryl, (8) aryl C₁₋₄ alkyl,or (9) aryloxy; R^(f) is (1) hydrogen, (2) Cl₁₋₁₀ alkyl, (3) C₂₋₁₀alkenyl, (4) C₂₋₁₀ alkynyl, (5) aryl, (6) heteroaryl, (7) aryl C₁₋₁₅alkyl, (8) heteroaryl C₁₋₁₅ alkyl, (9) C₁₋₁₅ alkanoyl, (10) C₃₋₈cycloalkyl; wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkanoyland cycloalkyl are optionally substituted with one to four groupsselected from R^(e).

Also preferred are the analogs taught in U.S. Pat. No. 5,859,051. Theseanalogs have the following general formula:

In the embodiments according to Formula VII, as defined in U.S. Pat. No.5,859,051, R¹ is selected from the group consisting of H, C₁₋₆ alkyl,C₅₋₁₀ aryl, and C₅₋₁₀ heteroaryl, said alkyl, aryl and heteroaryloptionally substituted with 1 to 3 groups of R^(a); R¹ is selected froma group consisting of: H, C₁₋₁₅ alkyl, C₂₋₁₅ alkenyl, C₂₋₁₅ alkynyl andC₃₋₁₀ cycloalkyl, said alkyl, alkenyl, alkynyl, and cycloalkyloptionally substituted with 1 to 3 groups of R^(a); R³ is selected froma group consisting of: H, NHR¹, NHacyl, C₁₋₁₅ alkyl, C₃₋₁₀ cycloalkyl,C₂₋₁₅ alkenyl, C₁₋₁₅ alkoxy, CO₂ alkyl, OH, C₂₋₁₅ alkynyl, C₅₋₁₀ aryl,C₅₋₁₀ heteroaryl said alkyl, cycloalkyl, alkenyl, alkynyl, aryl andheteroaryl optionally substituted with 1 to 3 groups of R^(a); (Z—W—) isZ-CR⁶R⁷—, Z-CH.═CH—, or:

R⁸ is selected from the group consisting of CR⁶R⁷, O, NR⁶, and S(O)_(p);R⁶ and R⁷ are independently selected from the group consisting of H,C₁₋₆ alkyl; B is selected from the group consisting of: 1) a 5 or 6membered heterocycle containing 0 to 2 double bonds, and 1 heteroatomselected from the group consisting of O, S and N, heteroatom beingsubstituted at any position on the five or six membered heterocycle, theheterocycle being optionally unsubstituted or substituted with 1 to 3groups of R^(a); 2) a 5 or 6 membered carbocycle containing 0 to 2double bonds, the carbocycle optionally unsubstituted or substitutedwith 1 to 3 groups of R² at any position on the five or six memberedcarbocycle; and 3) a 5 or 6 membered heterocycle containing 0 to 2double bonds, and 3 heteroatoms selected from the group consisting of O,N, and S, which are substituted at any position on the five or sixmembered heterocycle, the heterocycle being optionally unsubstituted orsubstituted with 1 to 3 groups of R^(a); X¹ and X² are independentlyselected from a group consisting of: H, OH, C₁₋₁₅alkyl, C₂₋₁₅alkenyl,C₂₋₁₅ alkynyl, halo, OR³, ORCF₃, C₅₋₁₀ aryl, C₅-10 aralkyl, C₅₋₁₀heteroaryl and C₁₋₁₀ acyl, said alkyl, alkenyl, alkynyl, aryl andheteroaryl optionally substituted with 1 to 3 groups of R^(a); R^(a)represents a member selected from the group consisting of: halo, acyl,aryl, heteroaryl, CF₃, OCF₃, —O—, CN, NO₂, R³, OR³; SR³, N(OR), S(O)R³,SO₂R³, NR³R³, NR³COR³, NR³CO₂R³, NR³CON(R³)₂, NR³SO₂R³, COR³, CO₂R³,CON(R³)₂, SO₂N(R³)₂, OCON(R³)₂ said aryl and heteroaryl optionallysubstituted with 1 to 3 groups of halo or C₁₋₆ alkyl; Y is selected fromthe group consisting of: S(O)_(p), —CH₂—, —C(O)—, —C(O)NH—, —NR—, —O—,—SO₂NH—, —NHSO₂; Y¹ is selected from the group consisting of: O and C; Zis selected from the group consisting of: CO₂R³, R³CO₂R³, CONHSO₂Me,CONHSO₂, CONH₂ and 5-(1H-tetrazole); t and v are independently 0 or 1such that t+v=1 Q is a saturated or unsaturated straight chainhydrocarbon containing 2-4 carbon atoms and p is 0-2 with the provisowhen Z is CO₂R³ and B is a 5 membered heterocycle consisting of O, R³does not represent methyl.

Additional analogs suitable for practicing the inventive methods andcompositions include compounds taught in U.S. Pat. No. 5,847,008, U.S.Pat. No. 6,090,836 and U.S. Pat. No. 6,090,839, each of which is hereinincorporated by reference in its entirety to the extent not inconsistentwith the present disclosure.

Additionally a variety of suitable analogs are taught in U.S. Pat. No.6,274,608. Aryl and heteroaryl acetic acid and oxyacetic acid analogsare taught for instance in U.S. Pat. No. 6,160,000; substituted5-aryl-2,4-thiazolidinedione analogs are taught in U.S. Pat. No.6,200,998; other possible analogs such as polyunsaturated fatty acidsand eicosanoids are known (see for instance, Forman, B M, Chen, J, andEvans R M, PNAS 94:4312-4317. The compounds of these publications, whichare each herein incorporated by reference in their entirety to theextent not inconsistent with the present disclosure can be screened bythe methods provide below to provide compounds which are useful, forinstance, in reducing body fat, and body weight, modulating fatcatabolism, and reducing appetite according to the present disclosure.

Synthesis of Fatty Acid Alkanolamides

Compounds useful in practicing the present invention are readilysynthesized and purified using methods recognized in the art. In anexemplary synthetic scheme (Scheme 1), a carboxylic acid and anaminoalcohol (or an O-protected derivative thereof) are reacted in a thepresence of a dehydrating agent, e.g., dicyclohexylcarbodiimide, in anappropriate solvent. The fatty acid alkanol amide is isolated by methodssuch as extraction, crystallization, precipitation, chromatography andthe like. If the final product is the O-protected adduct, it isdeprotected, typically by an art-recognized method, to afford a fattyacid adduct having a free hydroxyl group.

Those of skill in the art will recognize that many variants on thescheme set forth above are available. For example, an activatedderivative, e.g, acyl halide, active ester, of the acid can be used.Similarly, a glycol (preferably mono O-protected) can be substituted forthe amino alcohol, resulting in an ester linkage between the twoconstituents of the molecule.

Reverse esters and reverse amides are also readily synthesized byart-recognized methods. For example, a hydroxycarboxylic acid is reactedwith an amine or hydroxy derivative of a long chain alkyl (i.e., C₄-C₂₂)in the presence of a dehydrating agent. In certain reaction pathways, itis desirable to protect the hydroxyl moiety of the hydroxycarboxylicacid.

Ethers and mercaptans are prepared by methods well-known to those ofskill in the art, e.g., Williamson synthesis. For example, a long chainalkyl alcohol or thiol is deprotonated by a base, e.g, NaH, and areactive alcohol derivative, e.g., a halo, tosyl, mesyl alcohol, or aprotected derivative thereof is reacted with the resulting anion to formthe ester or merptan.

The above-recited methods and variations thereof can be found in, forexample, RECENT DEVELOPMENTS IN THE Synthesis OF FATTY ACID DERIVATIVES,Knothe G, ed., Amer. Oil Chemists Society 1999; COMPREHENSIVE NATURALPRODUCTS CHEMISTRY AND O THER SECONDARY METABOLITES INCLUDING FATTYACIDS AND THEIR DERIVATIVES, Nakanishi K, ed., Pergamon Press, 1999;ORGANIC SYNTHESIS COLLECTED VOLUMES I-V, John Wiley and Sons; COMPENDIUMOF ORGANIC SYNTHETIC METHODS, Volumes 1-6, Wiley Interscience 1984;ORGANIC FUNCTIONAL GROUP PREPARATION, Volumes I-III, Academic Press Ltd.1983; Greene T, PROTECTING GROUPS IN ORGANIC SYNTHESIS, 2d ed., WileyInterscience 1991.

Methods of use, Pharmaceutical Compositions, and their Administration

Methods of Use

The compounds, compositions and methods of the invention (e.g., fattyacid alkanolamides, fatty acid ethanolamide compounds, analogs, andhomologues) are used to reduce body fat and or body weight in mammals,including dogs, cats, and especially humans. The weight loss may be foraesthetic or therapeutic purposes. The compounds may also be used toreduce appetite or induce hypophagia.

The compounds, compositions, and methods of the invention are used toprevent weight gain or body fat increases in individuals within a normalweight range. The compounds may be used in otherwise healthy individualswho are not otherwise in need of any pharmaceutical intervention fordiseases related to diabetes or hyperlipidemia or cancer. In someembodiments, the individuals to be treated are free of diseases relatedto disturbances in sugar or lipid levels or metabolism or free of riskfactors for cardiovascular and cerebrovascular disease. The individualsmay be non-diabetic and have blood sugar levels in the normal range. Theindividuals may also have blood lipids (e.g., cholesterol) ortriglyceride levels in the normal range. The individuals may be free ofatherosclerosis. The individuals may be free of other conditions such ascancer or other tumors, disorders involving insulin resistance, SyndromeX, and pancreatitis.

In other embodiments, the subjects are overweight or obese persons inneed of body fat and/or body weight reduction. In these embodiments, themethods, compounds, and compositions of the invention can beadministered to promote weight loss and also to prevent weight gain oncea body weight within the normal range for a person of that sex and ageand height has been achieved. The compounds may be used in otherwisehealthy individuals who are not in need of any pharmaceutical treatmentof a disorder related to diabetes, hyperlipidemia, or cancer. Theindividuals may also otherwise free of risk factors for cardiovascularand cerebrovascular diseases. In some embodiments, the individuals to betreated are free of diseases related to sugar (e.g., glucose) or lipidmetabolism. The individuals may be non-diabetic and have blood sugarlevels in the normal range. The individuals may also have blood lipids(e.g., cholesterol, HDL, LDL, total cholesterol) or triglyceride levelsin the normal range. The individuals may not need to be in treatment foratherosclerosis.

The compounds methods, and compositions of the invention may also beadministered to suppress appetite in mammals, including cats, dogs, andhumans. In some embodiments, the compounds may be used in otherwisehealthy individuals who are not in need of pharmaceutical interventionsfor any disease. In some embodiments, the individuals do not needpreventive or ameliorative therapy for diseases, including cancer,diabetes, or hyperlipidemia. In some embodiments, the individuals to betreated are free of diseases related to abnormal sugar or lipid levels.In other embodiments the individuals may be free of risk factors forcardiovascular or cerebrovascular disease. The individuals may benon-diabetic and have blood sugar levels in the normal range. Theindividuals may also have blood lipids (e.g., cholesterol) ortriglyceride levels in the normal range. The individuals may be free ofatherosclerosis.

The compounds methods, and compositions of the invention may also beadministered to modulate fat metabolism (e.g., increase fat catabolism)in mammals, including cats, dogs, and humans. In some embodiments, thecompounds may be used to reduce appetite in otherwise healthyindividuals. In some embodiments, the individuals to be treated are freeof diseases related to sugar or lipid metabolism (e.g., diabetes,hypercholesterolemia, low HDL levels or high LDL levels). Theindividuals may be non-diabetic and have blood sugar levels in thenormal range. The individuals may also have blood lipids (e.g.,cholesterol) or triglyceride levels in the normal range. The individualsmay be free of atherosclerosis.

Treatment with the compounds and compositions of the invention may befor a period predetermined by the degree or amount of weight loss hasbeen accomplished or when the individual achieves a BMI within thenormal range. Treatment with the compounds and compositions of theinvention may be reduced once a predetermined degree or amount of weightloss has been accomplished or when the individual achieves a BMI withinthe normal range

The compounds and compositions of the invention may be administeredsolely for the purposes of reducing body fat or reducing appetite.

Pharmaceutical Compositions.

Another aspect of the present invention provides pharmaceuticalcompositions which comprise compounds of the invention and apharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention comprise acompound of the instant invention as an active ingredient or apharmaceutically acceptable salt thereof, and may also contain apharmaceutically acceptable carrier and optionally other therapeuticingredients.

The compositions include compositions suitable for oral, rectal,topical, parenteral (including subcutaneous, intramuscular, andintravenous), ocular (ophthalmic), pulmonary (nasal or buccalinhalation), or nasal administration, although the most suitable routein any given case will depend in part on the nature and severity of theconditions being treated and on the nature of the active ingredient. Anexemplary route of administration is the oral route. The compositionsmay be conveniently presented in unit dosage form and prepared by any ofthe methods well-known in the art of pharmacy.

In practical use, the compounds of the invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). In preparing the compositions for oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like in the case of oral liquidpreparations, such as, for example, suspensions, elixirs and solutions;or carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like in the case of oral solid preparations such as, forexample, powders, hard and soft capsules and tablets, with the solidoral preparations being preferred over the liquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or nonaqueous techniques. Suchcompositions and preparations can contain at least 0.1 percent of activecompound. The percentage of active compound in these compositions may,of course, be varied and may conveniently be between about 2 percent toabout 60 percent of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that atherapeutically effective dosage will be obtained. The active compoundscan also be administered intranasally as, for example, liquid drops orspray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as sucrose, lactose or saccharin. When a dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor. To prevent breakdown during transit through the upperportion of the GI tract, the composition may be an enteric coatedformulation.

Administration

The compounds of the invention may also be administered parenterally.Solutions or suspensions of these active compounds can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The compounds of the invention can be effective over a wide dosagerange. For example, in the treatment of adult humans, dosages from about10 to about 1000 mg, about 100 to about 500 mg or about 1 to about 100mg may be needed. Doses of the 0.05 to about 100 mg, and more preferablyfrom about 0.1 to about 100 mg, per day may be used. A most preferabledosage is about 0.1 mg to about 70 mg per day. In choosing a regimen forpatients, it may frequently be necessary to begin with a dosage of fromabout 2 to about 70 mg per day and when the condition is under controlto reduce the dosage as low as from about 0.1 to about 10 mg per day.For example, in the treatment of adult humans, dosages from about 0.05to about 100 mg, preferably from about 0.1 to about 100 mg, per day maybe used. The exact dosage will depend upon the mode of administration,on the therapy desired, form in which administered, the subject to betreated and the body weight of the subject to be treated, and thepreference and experience of the physician or veterinarian in charge.

Generally, the compounds of the present invention can be dispensed inunit dosage form comprising preferably from about 0.1 to about 100 mg ofactive ingredient together with a pharmaceutically acceptable carrierper unit dosage. Usually, dosage forms suitable for oral, nasal,pulmonary or transdermal administration comprise from about 0.001 mg toabout 100 mg, preferably from about 0.01 mg to about 50 mg of thecompounds admixed with a pharmaceutically acceptable carrier or diluent.For storage and use, these preparations preferably contain apreservative to prevent the growth of microorganisms.

Administration of an appropriate amount the candidate compound may be byany means known in the art such as, for example, oral or rectal,parenteral, intraperitoneal, intravenous, subcutaneous, subdermal,intranasal, orintramuscular. In some embodiments, administration istransdermal. An appropriate amount or dose of the candidate compound maybe determined empirically as is known in the art. An appropriate ortherapeutic amount is an amount sufficient to effect a loss of body fator a loss in body weight in the animal over time. The candidate compoundcan be administered as often as required to effect a loss of body fat orloss in body weight, for example, hourly, every six, eight, twelve, oreighteen hours, daily, or weekly

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

With respect to transdermal routes of administration, methods fortransdermal administration of drugs are disclosed in Remington'sPharmaceutical Sciences, 17th Edition, (Gennaro et al. Eds., MackPublishing Co., 1985). Dermal or skin patches are a preferred means fortransdermal delivery of the compounds of the invention. Patchespreferably provide an absorption enhancer such as DMSO to increase theabsorption of the compounds. Other methods for transdermal drug deliveryare disclosed in U.S. Pat. Nos. 5,962,012, 6,261,595, and 6,261,595.Each of which is incorporated by reference in its entirety.

Preferred patches include those that control the rate of drug deliveryto the skin. Patches may provide a variety of dosing systems including areservoir system or a monolithic system, respectively. The reservoirdesign may, for example, have four layers: the adhesive layer thatdirectly contacts the skin, the control membrane, which controls thediffusion of drug molecules, the reservoir of drug molecules, and awater-resistant backing. Such a design delivers uniform amounts of thedrug over a specified time period, the rate of delivery has to be lessthan the saturation limit of different types of skin.

The monolithic design, for example, typically has only three layers: theadhesive layer, a polymer matrix containing the compound, and awater-proof backing. This design brings a saturating amount of drug tothe skin. Thereby, delivery is controlled by the skin. As the drugamount decreases in the patch to below the saturating level, thedelivery rate falls.

Compounds of the invention may be used in combination with othercompounds of the invention or with other drugs that may also be usefulin dieting or the treatment, prevention, suppression or amelioration ofbody fat. Such other drugs may be administered, by a route and in anamount commonly used therefor, contemporaneously or sequentially with acompound of the invention. When a compound of the invention is usedcontemporaneously with one or more other drugs, a pharmaceuticalcomposition in unit dosage form containing such other drugs and thecompound is preferred. When used in combination with one or more otheractive ingredients, the compound of the present invention and the otheractive ingredients may be used in lower doses than when each is usedsingly. Accordingly, the pharmaceutical compositions of the presentinvention include those that contain one or more other activeingredients, in addition to the compounds disclosed above.

IDENTIFICATION OF COMPOUNDS OF THE INVENTION

Candidate compounds, such as disclosed above, can be screened by avariety of means known in the art. Body fat reducing compounds, forinstance, can be identified in vivo using animal bioassay techniquesknown to those of ordinary skill in the art. Test compounds andappropriate vehicle or caloric controls can be administered by any of anumber of routes (e.g., the oral route, a parenteral route) toexperimental subjects and the weight of the subjects can be monitoredover the course of therapy. The experimental subjects are humans or testanimals (e.g., rats, mice).

The effect of the compound on appetite or in inducing hypophagia orreduced food intake can be assessed, for instance, by monitoring thefood consumption of the test subjects (e.g., measuring the amount eatenor not eaten by a subject in terms of food weight or caloric content).The effect of the compounds on appetite can also be assessed bysubjective means including questionnaires as to appetite or foodcravings levels by human subjects. The effect of the test compounds onlipid metabolism can be assessed by monitoring blood lipids and fattyacid oxidation. The techniques for these assessments are well known tothose of ordinary skill in the art. The studies may be acute, subacute,chronic, or subchronic with respect to the duration of administrationand or follow-up of the effects of the administration.

Body fat reduction can be determined, for instance, by directlymeasuring changes in body fat of the animal or by measuring changes inthe body weight of the animal. The animal may selected from the groupconsisting of a mouse, a rat, a guinea pig, or a rabbit. The animal mayalso be an ob/ob mouse, a db/db mouse, or a Zucker rat or other animalmodel for a weight-associated disease. Clinical studies in humans mayalso be conducted.

Combinatorial Chemical Libraries

Recently, attention has focused on the use of combinatorial chemicallibraries to assist in the generation of new chemical compound leads. Acombinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks called amino acids in every possible way for a given compoundlength (i.e., the number of amino acids in a polypeptide compound).Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks. For example, onecommentator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. J. Med. Chem. 37(9):1233 (1994)).

Preparation and screening of combinatorial chemical libraries are wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, p benzodiazepines (U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al. PNAS USA 90: 6909 (1993)), analogous organicsyntheses of small compound libraries (Chen et al.) J. Amer. Chem. Soc.116: 2661 (1994), oligocarbamates (Cho, et al., Science 261: 1303(1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem.59: 658 (1994)), and small organic molecule libraries (see, e.g.,benzodiazepines (Baum C&EN, January 18, page 33 (1993)), thiazolidinonesand metathiazanones (U.S. Pat. No. 5,549,974), pyrrolidines (U.S. Pat.Nos. 5,525,735 and 5,519,134), benzodiazepines (U.S. Pat. No.5,288,514), and the like.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,HewlettPackard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices so that they can operate as discussedherein will be apparent to persons skilled in the relevant art. Inaddition, numerous combinatorial libraries are themselves commerciallyavailable (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru,Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

High Throughput Assays of Chemical Libraries

The assays for compounds described herein are amenable to highthroughput screening. Preferred assays thus detect activation oftranscription (i.e., activation of mRNA production) by the testcompound(s), activation of protein expression by the test compound(s),or binding to the gene product (e.g., expressed protein) by the testcompound(s); or effects on fatty acid modulation as described below.

High throughput assays for the presence, absence, or quantification ofparticular protein products or binding assays are well known to those ofskill in the art. Thus, for example, U.S. Pat. No. 5,559,410 discloseshigh throughput screening methods for proteins, and U.S. Pat. Nos.5,576,220 and 5,541,061 disclose high throughput methods of screeningfor ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

Determining Whether Compounds Affect Food Intake, Body Weight, Body Fat,Appetite, Food Seeking Behavior, or Modulate Fatty Acid Oxidation

Compounds of the invention can be administered to an animal to determinewhether they affect food intake and body weight, body fat, appetite,food seeking behavior, or modulate modulator fatty acid oxidation.

Animals can be, for example, obese or normal guinea pigs, rats, mice, orrabbits. Suitable rats include, for example, Zucker rats. Suitable miceinclude, for example, normal mice, ALS/LtJ, C3.SWH-H^(2b)/SnJ,(NON/LtJ×NZO/H1J)F1, NZO/H1J, ALR/LtJ, NON/Ltj, KK.Cg-AALR/LtJ, NON/LtJ,KK.Cg-A^(y)/J, B6.HRS(BKS)-Cpe^(fat)/+, B6.129P2-Gck^(tm/Efr),B6.V-Lep^(ob), BKS.Cg-m+/+Lep^(rd)b, and C57BL/6J with Diet InducedObesity.

Administration of an appropriate amount the candidate compound may be byany means known in the art such as, for example, oral or rectal,parenteral such as, for example, intraperitoneal, intravenous,subcutaneous, subdermal, intranasal, or intramuscular. Preferablyadministration may be intraperitoneal or oral. An appropriate effectiveamount of the candidate compound may be determined empirically as isknown in the art. An appropriate effective amount may be an amountsufficient to effect a loss of body fat or a loss in body weight orreduction in food consumption in the animal over time. The candidatecompound can be administered as often as required to effect a loss ofbody fat or loss in body weight, for example, hourly, every six, eight,twelve, or eighteen hours, daily, or weekly.

Formulations suitable for oral administration include (a) liquidsolutions, such as an effective amount of the candidate compoundsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms includeone or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates,corn starch, potato starch, microcrystalline cellulose, gelatin,colloidal silicon dioxide, talc, magnesium stearate, stearic acid, andother excipients, colorants, fillers, binders, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, dyes,disintegrating agents, and pharmaceutically compatible carriers. Lozengeforms can comprise the active ingredient in a flavor, e.g., sucrose, aswell as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, carriersknown in the art.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.Formulations suitable for parenteral administration, include, forexample, aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives.

The dose administered to the animal is sufficient to effect a change inbody weight, body fat, and/or fatty acid oxidation over time. Such adose can be determined according to the efficacy of the particularcandidate compound employed and the condition of the animal, as well asthe body weight or surface area of the animal. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a candidate compound;the LD₅₀ of the candidate compound; and the side-effects of thecandidate compound at various concentrations. In general, the dose willrange from 0.1-50 mg per kg, preferably 1-25 mg per kg, most preferably1-20 mg per kg body weight. The determination of dose responserelationships is well known to one of ordinary skill in the art.

Body Fat Reduction

Body weight reduction is typically determined by direct measurements ofthe change in body fat or by loss of body weight. Body fat and bodyweight of the animals is determined before, during, and after theadministration of the candidate compound. Changes in body fat remeasured by any means known in the art such as, for example, fat foldmeasurements with calipers, bioelectrical impedance, hydrostaticweighing, or dual x-ray absorbiometry. Preferably animals demonstrate atleast 2%, 5%, 8%, or 10% loss of body fat. Changes in body weight can bemeasured by any means known in the art such as, for example, on aportable scale, on a digital scale, on a balance scale, on a floorscale, or a table scale. Preferably animals demonstrate at least 2%, 5%,10%, or 15% loss of body weight. Body weight reduction is measuredbefore administration of the candidate compound and at regular intervalsduring and after treatment. Preferably, body weight is measured every 5days, more preferably every 4 days, even more preferably every 3 days,yet more preferably every 2 days, most preferably every day.

Changes in Fatty Acid Metabolism

Changes in fatty acid metabolism can be measured, for instance, bylooking at fatty acid oxidation in cells from major fat burning tissuessuch as, for example, liver (Beynen, et al. Diabetes 28:828 (1979)),muscle (Chiasson Lab. Anat. of Rat, (1980)), heart (Flink, et al. J.Biol. Chem. 267: 9917 (1992)), and adipocytes (Rodbell J. Biol. Chem.239: 375 (1964)), Cells may be from primary cultures or from cell lines.Cells may be prepared for primary cultures by any means known in the artincluding, for example, enzymatic digestion and dissection. Suitablecell lines are known to those in the art. Suitable hepatocyte lines are,for example, Fao, MH1C1, H-4-II-E, H4TG, H4-II-E-C3, McA-RH7777,McA-RH8994, NI-S1 Fudr, N1-S1, ARL-6, Hepa 1-6, Hepa-1c1c7, BpRc1, taoBpRc1, NCTC clone 1469, PLC/PRF/5, Hep 3B2.1-7 [Hep 3B], Hep G2 [HepG2],SK-HEP-1, WCH-17. Suitable skeletal muscle cell lines are, for example,L6, L8, C8, NOR-10, BLO-11, BC3H1, G-7, G-8, C2C12, P19, Sol8, SJRH30[RMS 13], QM7. Suitable cardiac cell lines are, for example, H9c2(2-1),P19, CCD-32Lu, CCD-32Sk, Girardi, FBHE. Suitable adipocyte lines are,for example, NCTC clone 929 [derivative of Strain L; L-929; L cell],NCTC 2071, L-M, L-M(TK-) [LMTK-; LM(tk-)], A9 (APRT and HPRT negativederivative of Strain L), NCTC clone 2472, NCTC clone 2555, 3T3-L1, J26,J27-neo, J27-B7, MTKP 97-12 pMp97b [TKMp97-12], L-NGC-5HT2, Ltk-11,L-alpha-1b, L-alpha-2A, L-alpha-2C, B82.

The rate of fatty acid oxidation may be measured by ¹⁴C-oleate oxidationto ketone bodies (Guzmán and Geelen Biochem. J. 287:487 (1982)) and/or¹⁴C-oleate oxidation to CO₂ (Fruebis PNAS 98:2005 (2001); Blazquez etal. J. Neurochem 71: 1597 (1998)). Lypolysis may be measured by fattyacid or glycerol release by using appropriate labeled precursors orspectrophotometric assays (Serradeil-Le Gal FEBS Lett 475: 150 (2000)).For analysis of ¹⁴C-oleate oxidation to ketone bodies, freshly isolatedcells or cultured cell lines can be incubated with ¹⁴C-oleic acid for anappropriate time, such as, for example, 30, 60, 90, 120, or 180 minutes.The amount of ¹⁴C radioactivity in the incubation medium can be measuredto determine their rate of oleate oxidation. Oleate oxidation can beexpressed as nmol oleate produced in x minutes per g cells. For analysisof lypolysis/glycerol release, freshly isolated cells or cultured cellslines can be washed then incubated for an appropriate time. The amountof glycerol released into the incubation media can provide an index forlypolysis.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnon-critical parameters which could be changed or modified to yieldessentially similar results.

Example 1 Synthesis of Fatty Acid Ethanolamide Compounds, Homologues andAnalogs

Methods for the formation of fatty acid ethanolamines from ethanolaminesand the corresponding fatty acyl are relatively straight forward andknown to one of ordinary skill in the art. For example, fatty acidethanolamides may be synthesized by reacting a fatty acid or fatty acidchloride with an aminoalcohol as described by Abadjj et al. (Abadji, V.,Lin, S. Y., Taha, G., Griffin, G., Stevenson, L. A., Pertwee, R. G. &Makriyannis, A. J. Med. Chem. 37, 1889-1893 (1994)). Fatty acids may beprepared similarly to the procedure of Serdarevich and Carroll(Serdarevich, B. & Carroll, K. K. J. Lipid Res. 7, 277-284 (1966)).Radioactively labeled fatty acid ethanolamides can be prepared byreaction with acyl chlorides (Nu-Check Prep, Elysian, Minn.) with[³H]ethanolamine (10-30 Ci/mmol; American Radiolabeled Chemicals, St.Louis) as described by Desamaud, F., Cadas, H. & Piomelli, D. (1995) J.Biol. Chem. 270, 6030-6035. Compounds can be purified by flash columnchromatography or HPLC. Compound identity can be established by use ofNMR and/or gas chromatography-mass spectrometry and thin layerchromatography.

Starting reagents and materials may be purchased from Avanti PolarLipids, Cayman Chemicals (Ann Arbor, Mich.), Nu-Check Prep, ResearchBiochemicals, or Sigma. Briefly, according to methods taught byGiuffrida, A. et al. (see Giuffrida, A. and Piomelli, D. in Lipid SecondMessengers (Laycock, S. G. and Rubin, R. P. Eds. pp. 113-133 CRC PressLLC, Boca Raton, Fla.) and Devane et al. (Devane W., Hanus, L. et al.Science 258, 1946-1949 (1992)), unlabeled or labeled fatty acylethanolamines can be synthesized by the reaction of the correspondingfatty acyl chlorides with unlabeled or labeled ethanolamine. The fattyacid chlorides can be dissolved in dichloromethane (10 mg/ml) andreacted with ethanolamine at −0.4° C. for 15 minutes. The reaction canbe quench by the addition of purified water. After vigorous stirring thephases are allowed to separate. The upper aqueous phase is discarded.The organic phase is washed twice with water. These washes remove theunreacted ethanolamine. This method provides a quantitative formation offatty acyl ethanolamines. The ethanolamines are concentrated to drynessunder a stream of nitrogen gas and can be reconstituted in an organicsolvent such as dichloromethane at a concentration of 20 mM. Theresulting fatty acyl ethanolamine solution can be stored at −20° C.until needed for use.

The chemistry of fatty acid carboxylic acid groups, primary andsecondary amines, and primary alcohol groups is well known to one ofordinary skill in the art. Fatty acid ethanolamides having a variety ofsubstituents on the ethanolamine portion thereof can be formed in manyways, but most preferably by starting with the corresponding substitutedethanolamine and fatty acid moieties. Such substituted ethanolamineswould include the alkyl aminoethanol ethers and acyl aminoethanol estersas well as secondary alkyl ethanol amines. Alternatively, the particularfatty acid ethanolamide can be synthesized from the corresponding fattyacid ethanolamide by the addition of the appropriate substituent groups.

Example 2 Methods for Screening Fatty Acid Ethanolamide (FAE) In Vivoand Other Compounds of the Invention

Animals. Male Wistar rats (200-350 g) were used. Procedures should metNIH guidelines detailed in the Guide for the Care and Use of LaboratoryAnimals, and the European Communities directive 86/609/EEC regulatinganimal research.

Chemicals. FAEs and [2H4] FAEs were synthesized in the laboratory(Giuffrida et al., “Lipid Second Messengers” (ed. Laychock, S. G. &Rubin, R. P.) 113-133 (CRC Press LLC, Boca Raton, Fla., 1998));1,2-dioleyl-sn-glycero-phosphoethanolamine-N-oleyl was purchased fromAvanti Polar Lipids (Alabaster, Ala.); SR141716A was provided by RBI(Natick, Mass.) as part of the Chemical Synthesis Program of the NIMH(N01 MH30003); SR144528 was a generous gift of Sanofi Recherche; allother drugs were from Tocris (Ballwin, Mo.) or Sigma (Saint Louis, Mo.).FAE were dissolved in dimethylsulphoxide (DMSO) and administered in 70%DMSO in sterile saline (acute treatments) or 5% Tween 80/5%propylenglycol in sterile saline (subchronic treatments) (1 ml per kg,i.p.). Capsaicin was administered in 10% Tween 80/10% ethanol/80%saline; SR141716A, SR144528, CCK-8 and CP-93129 in 5% Tween 80/5%propylenglycol/90% saline (1 ml per kg, i.p.).

Enzyme assays. In all biochemical experiments, rats were killed andtissues collected between 1400 and 1600 h, after varying periods of fooddeprivation. Microsome fractions were prepared as described (Désarnaudet al., J. Biol. Chem., 270:6030-6035 (1995)). NAT assays were performedusing 1,2-di[¹⁴C]palmityl-sn-glycerophosphocholine as a substrate (108mCi/mmol, Amersham, Piscataway, N.J.) (Cadas et al., H., J. Neurosci.,17:1226-1242 (1997)). FAAH assays were performed according to (Désarnaudet al., J. Biol. Chem., 270:6030-6035 (1995)), except that[³H]anandamide (arachidonyl-[113H]ethanolamide; 60 Ci/mmol; ARC, St.Louis, Mo.) was included as a substrate and radioactivity was measuredin the aqueous phase after chloroform extraction.

HPLC/MS analyses. Plasma was prepared from blood obtained by cardiacpuncture (Giuffrida et al., Anal. Biochem., 280:87-93 (2000)) and CSFwas collected from the cisterna magna using a 27G ½ needle(Precisionglide, USA). FAEs and NAPE were extracted from tissues withmethanol/chloroform and fractionated by column chromatography (Giuffridaet al., “Lipid Second Messengers” (ed. Laychock, S. G. & Rubin, R. P.)113-133 (CRC Press LLC, Boca Raton, Fla., 1998)). FAEs were quantifiedby HPLC/MS, using an isotope dilution method (Giuffrida et al., Anal.Biochem., 280:87-93 (2000)). Individual NAPE species were identified andquantified by HPLC/MS, using an external standard method (Calignano etal., Nature, 408:96-101 (2000)).

Blood chemistry. Plasma β-hydroxybutyrate and glycerol were measuredusing commercial kits (Sigma, St. Louis, Mo.). Plasma prolactin,corticosterone and luteinizing hormone were quantified byradioimmunoassay (Navarro et al., Neuroreport, 8:491-496 (1997)).

Feeding experiments. Acute experiments. Food intake was measured in 24-hfood-deprived rats (Navarro et al., J. Neurochem., 67:1982-1991 (1996)),administering drugs 15 min before food presentation. Subchronicexperiments. Ad libitum fed rats received vehicle injections for threedays. On day four, the animals were divided in two equal groups and gavethem daily injections of vehicle or OEA (5 mg per kg at 1900 h) for 7consecutive days, while measuring body weight, food intake and waterintake.

Conditioned taste aversion. Rats were water-deprived for 24 h and thenaccustomed to drink from a graded bottle during a 30-min test period forfour days. On day five, water was substituted with a 0.1% saccharinsolution and, 30 min later, the animals received injections of vehicle,OEA (20 mg per kg) or lithium chloride (0.4 M, 7.5 ml per kg). Duringthe following two days, water consumption was recorded over 30-min testperiods. The animals were then presented with water or saccharin, anddrinking measured.

Operant responses for food. Rats were trained to lever press for food ona fixed ratio 1 (FR1) schedule of reinforcement, while food-restrictedat 20 g of chow per rat per day (Rodríguez de Fonseca et al., ActaPharmacol. Sin., 20:1109-1114 (1999)). Once stable responding wasachieved, the animals were trained to acquire an FR5, time out 2-minschedule of food reinforcement and kept in limited access to food. Whena stable baseline was obtained, the animals were used to test theeffects of vehicle or OEA (1, 5 or 20 mg per kg) administered 15 minbefore lever presentation. Test duration was 60 min.

Other behavioral assays. The elevated plus maze test was conducted asdescribed (Navarro et al., Neuroreport, 8:491-496 (1997)) after theadministration of vehicle or OEA (20 mg per kg, i.p.). Horizontalactivity in an open field (Beltramo et al., J. Neurosci., 20:3401-3407(2000)) and pain threshold in the hot plate test (55° C.) (Beltramo etal., Science, 277:1094-1097 (1997)) were measured 15 min after injectionof vehicle or OEA (20 mg per kg). Rectal temperature was measured usinga digital thermometer (Martin-Calderón et al., Eur. J. Pharmacol.,344:77-86. (1998)).

In situ hybridization. Rats were accustomed to the handling andinjection procedure for five days. On day six, vehicle or drug OEA (10mg per kg, i.p.), or oleic acid (10 mg per kg) was administered, and therats killed 60 min later by decapitation under anesthesia. In situhybridization analyses were conducted using ³⁵S-labeled cRNA probes forc-fos (Guthrie et al., Proc. Natl. Acad. Sci. U.S.A., 90:3329-3333(1993)) and choline acetyl transferase (ChAT) (Lauterborn et al., BrainRes. Mol. Brain. Res., 17:59-69 (1993)). Average hybridization densitieswere determined from at least three tissue sections per rat. Statisticalsignificance was evaluated using one-way analysis of variance (ANOVA)followed by the Tukey-Kramer post-hoc test for paired comparisons.

Data analysis. Results are expressed as mean±s.e.m of n separateexperiments. The significance of differences among groups was evaluatedusing ANOVA followed by a Student-Newman-Keuls post hoc test, unlessindicated otherwise.

Example 3 Effects of Starvation on OEA and Other FAE Levels in the Rat

In one embodiment, the invention provides methods of treatment whereinindividuals needing to lose weight and/or body fat are tested for OEAlevels before and/or during fasting. Individuals with low levels of OEAprior to or in response to fasting are particularly then targeted forOEA treatment.

Rats were deprived of food while periodically measuring FAE levels incardiac blood by high-performance liquid chromatography (HPLC) coupledto electrospray mass spectrometry (MS). Plasma OEA remained at baselinelevels for the first 12 h of fasting, markedly increased at 18-24 h, andreturned to normal at 30 h (FIG. 1 a). No such effect was observedfollowing water deprivation (FIG. 1 b) or application of stressors suchas restraint immobilization and lipopolysaccharide (LPS) administration[in pmol per ml; 10.3±0.8; 60 min after a 15-min immobilization,8.4±1.6; 60 min after LPS injection (1 mg per kg), 7.0±0.7; n=6-9].Plasma PEA was not significantly affected by any of these treatments(data not shown), whereas anandamide decreased rapidly upon foodremoval, remaining lower than baseline for the entire duration of theexperiment (FIG. 1 d). Anandamide levels also declined afterimmobilization (in pmol per ml; control, 3.6±0.4; immobilization,1.1±0.5; n=7-8; P<0.01), LPS treatment (control, 2.0±0.5; LPS, 0.2±0.2;n 6; P<0.01) and, though not significantly, water deprivation (FIG. 1e). These results indicate that circulating OEA levels increasetransiently during starvation. This response is selective for OEA overanandamide and other FAEs, and coincides temporally with the rise inblood glycerol and β-hydroxybutyrate (Table 1), which signals the shiftof energy metabolism from carbohydrates to fatty acids as primary fuel(Cahill, G. F., Clin. Endocrinol. Metab., 5:397-415 (1976)).

TABLE 1 Plasma level of (β-hydroxybutyrate (β-HBA) and glycerol infasting rats. β-HBA Glycerol Free feeding 1.2 ± 0.4 4.6 ± 0.9  2 hfasted 1.2 ± 0.2 5.3 ± 0.6  4 h fasted 0.8 ± 0.1 9.1 ± 1.8  8 h fasted1.3 ± 0.2 6.3 ± 0.4 12 h fasted  4.6 ± 0.8* 7.6 ± 1.0 18 h fasted  6.8 ±0.4*  8.4 ± 0.4* 24 h fasted  9.1 ± 1.2*  8.4 ± 0.3* Concentrations areexpressed in mg per dl. *P < 0.05, n = 3 per group.

OEA levels in cerebrospinal fluid were not significantly affected byfood deprivation (FIG. 1 c), implying that the surge in plasma OEA mayoriginate outside the CNS. To test this hypothesis, the impact ofstarvation on OEA metabolism in various rat tissues was investigated.The biochemical route by which animal cells produce and degrade OEA andother FAEs is thought to comprise three key enzymatic steps. Calciumion-stimulated NAT activity transfers a fatty acid group from the sn-1position of a donor phospholipid to the primary amine ofphosphatidylethanolamine, producing NAPE2 (Schmid et al., Chem. Phys.Lipids, 80:133-142 (1996); Piomelli et al., Neurobiol. Dis., 5:462-473(1998)). Cleavage of the distal phosphodiester bond in NAPE by anunknown phospholipase D generates FAEs (Schmid et al., Chem. Phys.Lipids, 80:133-142 (1996); Piomelli et al., Neurobiol. Dis., 5:462-473(1998)), which are eventually broken down to fatty acid and ethanolamineby an intracellular fatty acid amide hydrolase (FAAH) (Schmid et al., J.Biol. Chem., 260:14145-14149 (1985); Cravatt et al., Nature, 384:83-87(1996)). Food deprivation (18 h) was accompanied by a marked increase inNAT activity in white adipose tissue (FIG. 2 a), but not in the brain,stomach or kidney (FIG. 2 b,d and data not shown). In liver, intestinesand skeletal muscle, NAT activity was reduced by fast (FIG. 2 c,d anddata not shown). These enzymatic changes were paralleled bycorresponding alterations in NAPE tissue content. Several molecularspecies of NAPE are present in rat tissues, including the OEA precursorsalk-1-palmitoenyl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-oleyl(NAPE 1; FIG. 3 a) andalk-1-palmityl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-oleyl(NAPE 2; FIG. 3 a); and the PEA precursoralk-1-palmityl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-palmityl(not shown). In agreement with NAT activity measurements, fooddeprivation increased NAPE content in fat, and decreased it in liver(FIG. 3 b,c).

Since NAPE biosynthesis and FAE formation are tightly coupled processes(Cadas et al., H., J. Neurosci., 17:1226-1242 (1997)), one might expectstarvation to augment the levels of OEA and other FAEs in adipose, butnot in other tissues. Accordingly, fat from starved rats contained moreOEA and PEA than did fat from free-feeding controls (FIG. 3 d and datanot shown), whereas no such difference was seen in the brain, stomach,and intestines (data not shown). Contrary to our expectation, however,the liver content of OEA and PEA was also higher in food-deprived thanin free-feeding rats (FIG. 3 d and data not shown). This discordance maybe due to an accumulation of FAEs by the liver, which is consistent withthe postulated roles of this organ in FAE recapture and metabolism(Bachur et al., J. Biol. Chem., 240:1019-1024 (1.965); Schmid et al., J.Biol. Chem., 260:14145-14149 (1985)).

The hydrolysis to fatty acid and ethanolamine, catalyzed by FAAH, is akey step in FAE degradation (Bachur et al., J. Biol. Chem.,240:1019-1024 (1965); Schmid et al., J. Biol. Chem., 260:14145-14149(1985); Cravatt et al., Nature, 384:83-87 (1996); Désarnaud et al., J.Biol. Chem., 270:6030-6035 (1995)). Food deprivation profoundly reducedFAAH activity in adipose membranes, but had no effect on FAAH activityin the brain, liver, stomach, intestines, kidney and skeletal muscle(FIG. 2 a-e and data not shown). Thus, food deprivation may increase thelevels of OEA and other FAEs in white fat in two synergistic ways, whichare mechanistically distinct from other reactions occurring duringlipolysis: stimulation of NAT activity may lead to increase thebiosynthesis of NAPE and FAEs, while inhibition of FAAH activity mayprolong the life span of newly synthesized FAEs. Although severaltissues may contribute to the normal levels of OEA in the bloodstream,the dynamic biochemical changes observed in fat underscore the crucialrole of this tissue in generating OEA during starvation.

Example 4 Suppression of Food Intake by OEA and Other FAEs

The effects of systemically administered OEA on food intake in rats canbe assessed using a 24 h fast. In this system, OEA caused a dose- andtime-dependent suppression of food intake (FIG. 4 a,b). To define theselectivity of this response, various OEA analogs were evaluated fortheir ability to produce hypophagia.

Anandamide and oleic acid had no effect.

Palmitylethanolamide was active but significantly less potent than OEA.

Elaidylethanolamide (an unnatural OEA analog) was similar in potency toOEA (FIG. 4 a).

These results indicate that OEA reduces eating in a structurallyselective manner and that other fatty acid ethanolamide-like compoundscan be identified for use according to the invention.

Example 5 Specificity Over Cannabinoid Receptor Activators

The molecular requisites for OEA hypophagia are distinct from thoseinvolved in the interaction of anandamide with its known cannabinoidtargets (Khanolkar et al., Life Sci., 65:607-616 (1999)). Cannabinoidreceptor antagonists did not affect OEA hypophagia in vivo, and OEA didnot displace cannabinoid binding to rat brain membranes in vitro. Thus,despite its structural and biogenetic relationships with anandamide, OEAdoes not depend on the endogenous cannabinoid system to produceanorexia.

Example 6 Sustained Body Weight Reduction

In some embodiments, the compounds of the instant invention provide fora sustained fat reduction or body weight reduction upon prolongedadministration to mammals. This effect is advantageous as a variety ofdrugs suppress eating after acute administration, but fail to do so whentreatment is prolonged (Blundell, J., Trends Pharmacol. Sci., 12:147-157(1991)).

OEA was subohronically administered to rats. Daily injections of OEA (5mg per kg, i.p.) for seven days resulted in a small, but significantdecrease in cumulative food intake (FIG. 5 a), which was accompanied bya profound inhibition of weight gain (FIG. 5 b, c). OEA did not affectwater intake (FIG. 5 d). The impact of OEA on body weight is onlypartially explained by its moderate reduction of food consumptionindicating that other factors, such as stimulation of energy expenditureor inhibition of energy accumulation, may contribute to this effect.

Example 7 FAE's May Have a Peripheral Site of Action

In one of its aspects, the invention provides compounds with aperipheral site of action. Such a site is advantageous in reducing thelikelihood of central nervous system side effects.

Though potent when administered peripherally, OEA was ineffective afterdirect injection into the brain ventricles (Table 2), suggesting thatthe primary sites of action of this compound might be located outsidethe CNS. As a further demonstration, sensory fibers in the vagus andother peripheral nerves were chemically destroyed by treating adult ratswith the neurotoxin, capsaicin (Kaneko et al., Am. J. Physiol.,275:G1056-G1062 (1998)). Capsaicin-treated rats failed to respond toperipherally administered cholecystokinin-8 (CCK-8) (FIG. 6, a,c), drankmore water than controls (FIG. 6 b,d) and lost the corneal chemosensoryreflex (data not shown), three indications that the neurotoxin haddestroyed sensory afferents (MacLean, D. B., Regul. Pept., 11:321-333(1985); Ritter et al., Am. J. Physiol., 248:R501-R504 (1985); Curtis etal., Am. J. Physiol., 272:R704-R709 (1997)). Treated animals also failedto respond to OEA (10 mg per kg, i.p.), but responded normally to thecompound CP-93129, which targets 5-HT1B receptors in the CNS (FIG. 6a,c) (Lee et al., Psychopharmacology, 136:304-307 (1998)). Thesefindings support the hypothesis that OEA causes hypophagia by acting ata peripheral site, and that sensory fibers are required for this effect.

TABLE 2 Effects of intracerebroventricular pranamide on food intake. 60min 120 min 240 min vehicle 5.8 ± 0.6 8.0 ± 0.5 9.5 ± 0.5 prana 0.4 μg4.8 ± 0.4 6.6 ± 0.4 8.4 ± 0.4 prana 2 μg 4.9 ± 0.4 6.6 ± 0.6 8.7 ± 0.5prana 10 μg 5.9 ± 0.2 8.1 ± 0.4 9.6 ± 0.7 Pranamide/OEA(prana, μg peranimal) or vehicle (DMSO, 5 μl) was administered to 24 h food-deprivedrats 15 min before food presentation, n = 12 per group.

The compounds of the invention may use peripheral sensory inputs tosuppress appetite. Peripheral sensory inputs related to appetitesuppression recruit several CNS structures, which include the nucleus ofthe solitary tract (NST) in the brainstem and the arcuate andparaventricular (PVN) nuclei in the hypothalamus (Schwartz et al.,Nature, 404:661-671 (2000)). To identify the brain pathways engagedduring OEA-induced hypophagia, mRNA levels for the activity regulatedgene c-fos (Curran et al., Oncogene, 2:79-84 (1987)) were mapped by insitu hybridization after systemic administration of OEA, oleic acid orvehicle. When compared to controls, OEA (10 mg per kg, i.p.) evoked ahighly localized increase in c-fos mRNA levels in the PVN, supraopticnucleus (FIG. 7 a) and NST (FIG. 7 c). This enhancement was specific tothese areas, insofar as c-fos expression in other brain regions was notsignificantly affected by OEA treatment (FIG. 7 b,d). The finding thatOEA stimulates c-fos mRNA expression in the NST (which processes vagalsensory inputs to the CNS) and the PVN (a primary site for theorchestration of central catabolic signals) (Schwartz et al., Nature,404:661-671 (2000)), is consistent with a physiological role for thislipid as a peripheral mediator of anorexia.

It is possible that OEA reduced eating by inducing a non-specific stateof behavioral suppression. If this is the case, OEA should causeconditioned taste aversion, which can be readily provoked in rats by anumber of noxious substances (Green et al., Science, 173:749-751(1971)), including lithium chloride (FIG. 4 c). However, a maximal doseof OEA (20 mg per kg, i.p.) had little effect in this assay (FIG. 4 c),suggesting that the compound may not be aversive. Several additionalobservations support the behavioral specificity of OEA. OEA did notalter water intake, body temperature, pain threshold (FIG. 4 d-f), oractivity of the hypothalamus-pituitary-adrenal (HPA) axis (Table 3).Moreover, OEA did not produce anxiety-like symptoms (FIG. 4 g) and,though it reduced motor activity and operant responses for food, it didso at a dose that was substantially higher than those required toproduce hypophagia (FIG. 4 h-i). This pharmacological profiledifferentiates OEA from other appetite suppressants such as amphetamineand glucagon-like peptide 1 (whose effects often include aversion,hyperactivity, anxiety and activation of the HPA axis) and from theendogenous cannabinoid anandamide (which stimulates food intake inpartially satiated animals, increases pain threshold, decreases bodytemperature and activates the HPA axis) (Pertwee, R. G., Exp. Opin.Invest. Drugs, 9:1553-1571 (2000)).

TABLE 3 Effects of OEA on plasma hormone levels. B PRL LH vehicle 212 ±24 10.8 ± 2.7 5.3 ± 0.9 prana 20 280 ± 61  8.2 ± 3.2 6.2 ± 1.5 In Table2, plasma corticosterone (B), prolactin (PRL) and luteinizing hormone(LH) levels were measured by radioimmunoassay in plasma samplescollected 60 min after injection of vehicle or pranamide (prana, in mgper kg, i.p.) and are expressed in ng per ml. n = 6-9 per group.

OEA elicits hypophagia at physiologically relevant doses. 1 hr afteradministration of a half-maximally effective dose (5 mg per kg, i.p.),circulating OEA levels (16.1±2.6 pmol per ml) were significantly higherthan baseline (10.1±1.1; P<0.05, Student's t test; n=5), but below thosemeasured in 18-h food-deprived animals (FIG. 1 a). Thus, theconcentrations reached by OEA in blood during starvation can besufficient to elicit notable behavioral responses.

Example 8 Identifying Body Fat Reducing Compounds of the Invention

The following example demonstrates how to identify appetite suppressorsusing OEA as a positive control. In particular, the synthesis of OEA,the measurement of body fat reduction and fatty acid oxidation arediscussed.

Synthesis of OEA.

Oleylchloride is purchased from Nu-Check Prep (Elysian, Minn.) orprepared following standard procedures. Oleylchloride is dissolved indichloromethane (10 mg/ml) and allowed to react with five equivalents ofethanolamine for 15 min. at 0-4° C. The reaction is stopped by theaddition of purified water. After vigorous stirring and phaseseparation, the upper aqueous phase is discarded and the organic phaseis washed twice with water to remove non-reacted ethanolamine. Theresulting OEA is concentrated to dryness under a N₂ stream,reconstituted in chloroform at 20 mM, and stored at −20° C. until use.

Measuring Body Fat Reduction Induced by Candidate Compounds

The ability of a compound to reduce body fat can be evaluated by anumber of methods. For example, appropriate amounts OEA and/or candidatecompounds are administered to rats via intraperitoneal injection. TheOEA and candidate compounds can be formulated in 70% DMSO in sterilesaline, 5% Tween 80/5% propylenglycol in sterile saline, or 10% Tween80/10% ethanol/80% saline. Five mg per kg of OEA can be used as thepositive control. Amounts of candidate compounds administered may range,for instance, from 1-25 mg per kg. Typically 1, 2, 5, 10, 15, and 20 mgper kg doses of each candidate compound can be administered to differentsets of rats to determine which dose is optimal. Injections may be given30 minutes before the animals' principal meal for 7-14 days.

The effect of the candidate compound on total body fat can be determinedby taking direct measurements of the rat's body fat using skin foldcalipers. Skin on the rats' backs, abdomen, chest, front and rear legscan be pinched with calipers to obtain measurements beforeadministration of OEA and/or candidate compounds and every 48 hoursduring and after administration of OEA and/or candidate compounds.Differences in measurements in at least two of the pinched sites reflectthe change in the rat's total body fat.

Measuring Fatty Acid Oxidation Induced by Candidate Compounds

Compounds can also be assayed for their effect on fatty acid metabolism.The effect of the candidate compound on fatty acid metabolism can bemeasured by measurements of fatty acid oxidation in primary cultures ofliver cells. Hepatocytes may be used to determine the rate of oleateoxidation to ketone bodies and carbon dioxide. Such cells can beisolated from adult rat liver by enzymatic digestion as described byBeynen et al. in Diabetes 28:828 (1979). Cells typically are cultured insuspension and incubated in Krebs-Henseleit's bicarbonate mediumsupplemented with bovine serum albumin and glucose as described byGuzmán & Geelen, Biochem. J. 287:487 (1992). The protein concentrationof the cultured cells can be determined and cells seeded in 2 ml mediaso that 4-6 mg protein per ml is present in the reaction mixture. Cellscan be incubated for 10 minutes at 37° C. with [¹⁴C]-oleic acid(Amersham), in the presence or absence of 10 μM OEA, reactions may bestopped with 200 μl 2M perchloric acid and acid-soluble productsextracted with chloroform/methanol/water (5:1:1, vol:vol:vol). Theaqueous phase can be removed and washed twice more. Proteinconcentration can be determined using a Lowry assay. The rate of oleateconversion into ketone bodies may be expressed as nmol of oleateoxidized per hour per mg protein and may be determined using liquidscintillation counting. Accordingly, OEA enhances oleate oxidation by21+−6% (n=4, p<0.01 vs. control incubations by the Student t test).

Example 9 Effect of OEA on Fatty Acid Metabolism

Oleoylethanolamide (OEA) decreases body weight not only by suppressingappetite, but also by possibly enhancing body fat catabolism. Theeffects of OEA on fatty acid oxidation in major body-fat burning tissues(soleus muscle, liver, cultured cardiac myocytes and astrocytes) wasexamined. OEA significantly stimulates fatty acid oxidation in primarycultures of liver, skeletal muscle (soleus) and heart cells, whereas ithas no effect in brain-derived astroglial cell cultures. In addition,OEA induces a significant mobilization of triacylglycerol stores fromprimary white adipose tissue cells. Table 4 details the methods andeffects of OEA on fatty acid oxidation in these cells.Structure-activity relationship experiments provide evidence that theeffect of OEA on skeletal muscle fatty acid oxidation is specific (FIG.8). Thus, the effects of OEA are mimicked by the hydrolysis-resistanthomologue methyl-OEA and -only partially—by palmitylethanolamide (PEA),but not by arachidonylethanolamide (AEA) or oleic acid (OA). In short,these results show that lipid oxidation and mobilization are enhanced byOEA, and that the effects of OEA are restricted to peripheral sites.

TABLE 4 Cell/tissue Hepatocyte Soleus muscle Cardiomyocyte AstrocyteAdipocyte Origin Adult rat liver Adult rat hind Newborn rat Newborn ratAdult rat limb heart brain cortex epididymus Isolation EnzymaticDissection Enzymatic Enzymatic Enzymatic procedure digestion (Chiasson,digestion (Flink digestion digestion (Beynen et al., 1980) et al., 1992)(McCarthy & (Rodbell, 1979) De Vellis, 1964) 1980) Type of Cell TissueCell monolayer Cell Cell culture suspension suspension monolayersuspension Incubation Krebs- Krebs-Henseleit High-glucose Hams Krebs-medium Henseleit Hepes plus DMEM plus F12/DMEM Henseleit bicarbonate BSAand BSA plus insulin, Hepes plus plus BSA and glucose (Wu et al.,transferrin, BSA and glucose (Fruebis et al., 2000) progesterone,glucose (Guzman & 2001) putrescine (Rodbell, Geelen, 1992) and selenite1965) (Blazquez et al., 1998) Metabolic [¹⁴C]oleate [¹⁴C]oleate[¹⁴C]oleate [¹⁴C]oleate Lypolysis parameter oxidation to oxidation tooxidation to oxidation to (glycerol ketone bodies CO₂ (Fruebis et CO₂(Blazquez ketone bodies release) (Guzman & al., 2001) et al., 1998)(Blazquez et (Serradeil- Geelen, 1992) al., 1998) Le Gal et al., 2000)Incubation 10 30 30 30 30 time (min) Stimulatory 21 ± 6 (n = 4) 36 ± 10(n = 4) 37 ± 9 (n = 3) 2 ± 6 (n = 3) 38 ± 16 (n = 3) effect of 10 μM OEA(%) Statistical P < 0.01 P < 0.01 P < 0.01 Non P < 0.01 significancesignificant vs. control

References cited: Beynen A C et al., Diabetes 28:828-835 (1979);Blazquez C et al., J Neurochem 71:1597-1606 (1998); Chiasson R B“Laboratory Anatomy of the White Rat” WCB, Dubuque, Iowa (1980); Funk IL et al., J Biol Chem 267:9917-9924 (1992); Fruebis J et al., Proc NatlAcad Sci USA 98:2005-2010 (2001); Guzman M et al., Biochem J 287:487-492(1992); McCarthy K D et al., J Cell Biol 85:890-902 (198Q); Rodbell M JBiol Chem 239:375-380 (1964); Rodbell M Ann NY Acad Sci 131:302-314(1965); Serradeil-Le Gal C et al., FEBS Left 475:150-156 (2000); Wu W etal., J Biol Chem 275:40133-40119 (2000).

Example 10 Role of Endogenous OEA in the Intestines

The impact of feeding on intestinal OEA biosynthesis was studied. Highperformance liquid chromatography/mass spectrometry analyses revealedthat small intestinal tissue from free-feeding rats contains substantialamounts of OEA (354±86 pmol per g, n=3). Intestinal OEA levels weremarkedly decreased after food deprivation, but returned to baselineafter refeeding. By contrast, no such changes were observed in stomach(in pmol per g; control, 210±20; starvation, 238±84;starvation/refeeding, 239±60, n=3). Variations in intestinal OEA levelswere accompanied by parallel alterations in NAT activity, whichparticipates in OEA formation, but not in fatty acid amide hydrolaseactivity, which catalyzes OEA hydrolysis. These findings suggest thatstarvation and feeding reciprocally regulate OEA biosynthesis in smallintestine. In agreement with an intra-abdominal source of OEA, plasmaOEA levels in starved rats were found to be higher in portal than incaval blood (in pmol per ml; porta, 14.6±1.8; cava, 10.3±2.8; n=5). Thecontribution of other intra-abdominal tissues to OEA formation cannot beexcluded at present. These results suggest many interventions to utilizethe OEA systems in feeding behavior. According to this model, foodintake may stimulate NAT activity enhancing OEA biosynthesis in thesmall intestine and possibly other intra-abdominal tissues. Newlyproduced OEA may activate local, sensory fibers, which may in turninhibit feeding by engaging brain structures such as the NST and PVN.

Our results reveal an unexpected role for OEA in the peripheralregulation of feeding, and provide a framework to develop novelmedicines for reducing body weight or body fat, for preventing bodyweight gain or body fat increase, for suppressing appetite or reducingfood seeking behavior, or food intake, and for the treating eatingdisorders, overweight, or obesity. These medicines would include notonly OEA analogues and homologues but also agents which controlling OEAlevels by acting upon the OEA formation and hydrolyzing systems andenzymes as disclosed above.

All publications and patent applications cited in this specification areherein incorporated by reference to the extent not inconsistent with thepresent disclosure as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of modulating lipid metabolism in a mammal in need thereof,said method comprising administering to said mammal a fatty acidalkanolamide, wherein said administering is in an effective amount tomodulate lipid metabolism.
 2. The method according to claim 1, whereinthe fatty acid alkanolamide is oleoylethanolamide.
 3. The method ofclaim 1, wherein the fatty acid alkanolamide comprises a fatty acidmoiety covalently bonded to an ethanolamine moiety via an amide linkage.4. The method of claim 3, wherein the fatty acid moiety ismonounsaturated or polyunsaturated.
 5. The method of claim 1, whereinthe administering is via a dermal patch.
 6. The method of claim 4,wherein the fatty acid moiety is oleic acid.
 7. The method of claim 3,wherein the fatty acid moiety has from 12 to 20 carbon atoms.
 8. Themethod of claim 3, wherein the fatty acid is selected from the groupconsisting of elaidic acid, palmitoleic acid, palmitic acid, linoleicacid, alpha-linolenic acid, and gamma-linolenic acid.
 9. (canceled) 10.The method of claim 3, wherein the hydroxy group of the ethanolaminemoiety is bound to a the carboxylate group of a lower (C₂-C₆) alkylcarboxylic acid to form the corresponding ester.
 11. (canceled)
 12. Themethod according to claim 1, wherein the mammal is human.
 13. The methodaccording to claim 1, wherein the fatty acid alkanolamide ispalmitoylethanolamide.
 14. (canceled)
 15. The method according to claim1, wherein said fatty acid alkanolamide is administered with apharmaceutically acceptable carrier by an oral, rectal, topical, orparenteral route.
 16. A method of reducing or controlling body fat orbody weight in a mammal in need thereof, said method comprisingadministering to said mammal, in an effective amount to reduce body fator body weight, a compound of the formula

or a pharmaceutically acceptable salt thereof, wherein n is from 0 to 5,the sum of a and b can be from 0 to 4; Z is a member selected from thegroup consisting of C(O)N(Ro); (Ro)NC(O); OC(O); (O)CO; O; NRo; and S;and wherein Ro and R2 are members independently selected from the groupconsisting of unsubstituted or unsubstituted alkyl, hydrogen, C₁-C₆alkyl, and lower (C₁-C₆) acyl, and wherein up to four hydrogen atoms ofthe fatty acid portion and alkanol portion thereof are substituted bymethyl or a double bond, and the bond between carbons c and d may beunsaturated or saturated.
 17. A method of claim 16, wherein saidcompound is of the formula:

or a pharmaceutically acceptable salt thereof, wherein n is from 0 to 4,the sum of a and b is from 1 to 3, and R¹ and R² are membersindependently selected from the group comprising hydrogen, C₁-C₆ alkyl,and lower (C₁-C₆) acyl, and wherein up to four hydrogen atoms of thefatty acid portion and alkanolamine portion thereof are substituted bymethyl or a double bond, and the bond between carbons c and d may beunsaturated or saturated. 18-25. (canceled)
 26. The method of claim 16,wherein the mammal is a human.
 27. The method of claim 16, wherein thecompound is according to one of the following formulae:

wherein n is from 1-5 and the sum of a and b is from 0 to 4; R² isselected from the group consisting of hydrogen, C₁-C₆ alkyl, and lower(C₁-C₆) acyl; and up to four hydrogen atoms of the fatty acid portionand alkanol portion thereof may also be substituted by methyl or adouble bond.
 28. A pharmaceutical composition comprising apharmaceutically acceptable excipient and a compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein n is from 0 to 5,the sum of a and b can be from 0 to 4; Z is a member selected from thegroup consisting of C(O)N(R^(o))—; —(R^(o))NC(O)—; —OC(O)—; —(O)CO—; O;NR^(o); and S; and wherein R¹ and R² are members independently selectedfrom the group consisting of unsubstituted or unsubstituted alkyl,hydrogen, C₁-C₆ alkyl, and lower (C₁-C₆) acyl, and wherein up to fourhydrogen atoms of the fatty acid portion and alkanol portion thereof aresubstituted by methyl or a double bond, and the bond between carbons cand d may be unsaturated or saturated.
 29. A pharmaceutical compositionof claim 28, wherein the compound is of the formula:

or a pharmaceutically acceptable salt of the compound, wherein n is from1 to 3, the sum of a and b is from 1 to 3, and R¹ and R² are membersindependently selected from the group consisting of hydrogen, C₁-C₆alkyl, and lower (C₁-C₆) acyl and wherein up to four hydrogen atoms ofthe fatty acid portion and alkanol portion thereof are substituted bymethyl or a double bond, and the bond between carbons c and d may beunsaturated or saturated. 30-48. (canceled)
 49. The method of claim 1,wherein the modulating increases fat and fatty acid catabolism in themammal.
 50. The method of claim 17, wherein the modulating increases fatand fatty acid catabolism in the mammal.